Recombinant raccoonpox virus and uses thereof as a vaccine in mammalian and avian species

ABSTRACT

The present invention provides a recombinant raccoonpox virus comprising a raccoonpox virus viral genome which contains a foreign DNA sequence inserted into a non-essential region within the HindIII “U” genomic region, the HindIII “M” genomic region, or HindIII “N” genomic region of the raccoonpox virus genome. The present invention provides a recombinant raccoonpox virus comprising a raccoonpox virus viral genome which contains a deletion in a raccoonpox virus host range gene of the viral genome. The present invention provides a homology vector for producing a recombinant raccoonpox virus by inserting a foreign DNA sequence into the raccoonpox virus genome. The present invention provides a recombinant raccoonpox virus which is useful as a vaccine in mammalian and avian species.

Within this application several publications are referenced by arabicnumerals within parentheses. Full citations for these references may befound at the end of the specification immediately preceding the SequenceListing. The disclosures of these publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art as known to those skilled therein asof the date of the invention described and claimed herein.

BACKGROUND OF THE INVENTION

Raccoonpox virus (RPV) belongs to the orthopoxvirus family, likevaccinia virus, but has an advantage in that it is naturally attenuatedand appears to be native to North America. Unlike swinepox virus whoseinfection in non-porcine species is non-productive, raccoonpox virusinfection is productive, but may not spread systemically like vacciniavirus.

Raccoonpox virus was originally isolated from the upper respiratorytract of 2 of 92 apparently healthy raccoons in 1962 in Maryland (1).Significant HI (hemagglutinating-inhibiting) antibody titers for RPVwere demonstrated in 23% [22/92] of raccoon sera indicating that RPV isindigenous in North America (2). Serological surveys testing sera from593 raccoons in Delaware demonstrated that 4% were positive for RPVneutralizing antibodies, 12% were positive by ELISA and 2.5% of the serawere positive by both tests (10). RPV is classified as a orthopoxvirusbased upon biological, serological and biophysical methods. RPV andvolepox are the only orthopoxviruses thought to be native to NorthAmerica. The Hind III DNA restriction map of RPV is the most divergentfrom other orthopoxvirus DNA maps, which are highly conserved,indicating that RPV has diverged from the other group members (4,6). Of29 HindIII fragments from the RPV genome, none comigrated with HindIIIfragments that were common to other examined orthopoxvirus DNA. RPV isnaturally attenuated (10-100×) and avirulent compared with wild typevaccinia virus. There are four published RPV DNA sequences which includea Sal I end fragment (2.2 kb) (5), a hemagglutinin gene (1.5 kb) (7), aprotein phosphatase sequence (0.5 kb; H1L ) (15), and the TK gene (834bp) (16). RPV TK shows 87% amino acid homology to TK of vaccinia and84.3% nucleotide homology. RPV HA shows 53% amino acid homology withvaccinia HA and 66% nucleotide homology. No other information regardingthe genomic organization of RPV is known.

To date, recombinant raccoon poxviruses have been generated by insertionof foreign genes such as rabies virus glycoprotein into the TK locus ofraccoon poxvirus (7-14). RPV recombinants expressing foreign viralantigens have been shown to elicit host protective immune responses innon-raccoon species including dogs, cats, and sheep with no negativeeffects observed.

The present invention provides a recombinant raccoon poxvirus comprisinga foreign DNA inserted into the HindIII M region, HindIII N region orHindIII U region of the raccoonpox virus genome. The recombinant raccoonpoxvirus is useful as a vaccine in mammalian and avian species. Prior tothe present invention, it was unknown whether non-essential regionsexisted in the HindIII M, HindIII N, or HindIII U regions, and whether astable recombinant raccoonpox virus could be isolated with foreign DNAinsertions into these regions.

SUMMARY OF THE INVENTION

The present invention provides a recombinant raccoonpox virus comprisinga raccoonpox virus viral genome which contains a foreign DNA sequenceinserted into a non-essential region within the HindIII “U” genomicregion of the raccoonpox region of the raccoonpox virus genome.

The present invention provides a recombinant swinepox virus comprising aswinepox virus genome which contains a foreign DNA sequence insertedinto a non-essential region of the raccoonpox virus genome, wherein theforeign DNA sequence is a host range gene selected from the groupconsisting of raccoonpox virus K1L and raccoonpox virus C7L.

The present invention provides a recombinant raccoonpox virus comprisinga raccoonpox virus viral genome which contains a foreign virus viralgenome which contains a foreign DNA sequence inserted into anon-essential region within the HindIII “N” genomic region of theraccoonpox virus genome.

The present invention provides a recombinant raccoonpox virus comprisinga raccoonpox virus viral genome which contains a foreign DNA sequenceinserted into a non-essential region within the HindIII “M” genomicregion of the raccoonpox virus genome.

The present invention provides a recombinant raccoonpox virus comprisinga raccoonpox virus viral genome which contains a deletion in araccoonpox virus host range gene of the viral genome, wherein theraccoonpox virus host range gene is selected from the group consistingof C7L, C6L, C5L, C4L, C3L, C2L, C1L, N1L, N2L, M1L, M2L, and K1L.

The present invention provides a homology vector for producing arecombinant raccoonpox virus by inserting a foreign DNA sequence intothe raccoonpox virus genome which comprises a double-stranded DNAmolecule consisting of double-stranded foreign DNA sequence encoding anantigenic polypeptide derived from an animal pathogen. Located at oneend of the foreign DNA sequence, is double-stranded feline virus genomicDNA homologous to the genomic DNA located at one side of a non-essentialsite of the raccoonpox viral genomic DNA. Located at the other end ofthe foreign DNA sequence, is double-stranded raccoonpox virus genomicDNA homologous to the genomic DNA located at the other side of the samesite.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Restriction endonuclease map of raccoonpox virus HindIII “U”genomic fragment (approximately 2055 base pairs) (SEQ ID NO. 1). Twoopen reading frames (ORFS) are the O1L vaccinia homologue (SEQ ID NO. 3)and the E11L vaccinia homologue (SEQ ID NO. 2). (17) These two ORFs arenon-essential for viral replication and suitable for insertion offoreign DNA. Restriction endonuclease sites in the RPV HindIII U genomicfragment are shown as suitable insertion sites. Examples of recombinantraccoonpox of the present invention have foreign DNA inserted into theXbaI site at nucleotide 1065 of the HindIII U fragment.

FIG. 2: Restriction endonuclease map of raccoonpox virus HindIII “W”,“T”, and “P”, genomic fragments (approximately 8300 base pairs) (SEQ IDNO. 5, 12). At least seven open reading frames (ORFs) within this regionare the N1L vaccinia homologue (SEQ ID NO. 6), N2L vaccinia homologue(SEQ ID NO. 7), M1L vaccinia homologue (SEQ ID NO. 8), M2L vacciniahomologue (SEQ ID NO. 9), K1L vaccinia homologue (SEQ ID NO. 10), K2Lvaccinia homologue (SEQ ID NO. 11), and the K7R vaccinia homologue (SEQID NO. 13). (17) These seven ORFs are non-essential for viralreplication and suitable for insertion of foreign DNA. Restrictionendonuclease sites in the RPV HindIII W, T and P genomic fragments areshown as suitable insertion sites.

FIG. 3: Restriction endonuclease map of raccoonpox virus HindIII “N”genomic fragment (approximately 5,100 base pairs). At least two openreading frames (ORFs) are the D1L cowpox virus homologue (18) and theB22R variola virus homologue. These two ORFs are non-essential for viralreplication and suitable for insertion of foreign DNA. Restrictionendonuclease sites in the RPV HindIII N genomic fragment are shown assuitable insertion sites. Examples of recombinant raccoonpox of thepresent invention have foreign DNA inserted into the EcoRV site atapproximately nucleotide 1500 (S-RPV-009) or SnaBI site at approximatelynucleotide 2200 (S-RPV-008) of the HindIII U fragment.

FIG. 4: Restriction endonuclease map of raccoonpox virus HindIII “M”genomic fragment (approximately 6,000 base pairs). At least three openreading frames (ORFs) within this region are the D1L cowpox virushomologue (18) the C17L/B23R vaccinia virus homologue (17) and the D1Lvariola virus homologue (17). These three ORFs are non-essential forviral replication and suitable for insertion of foreign DNA. Restrictionendonuclease sites in the RPV HindIII M genomic fragment are shown assuitable insertion sites. Examples of recombinant raccoonpox of thepresent invention have foreign DNA inserted into the HpaI site atapproximately nucleotide 4100 (S-RPV-010) of the HindIII M fragment.

FIG. 5: DNA sequence of the synthetic pox viral promoter LP2EP2 andposition of unique EcoRI and BamHI sites (SEQ ID NO. 28) for insertionof foreign DNA under the control of the LP2EP2 promoter in homologyvector 919-16.12. The calorimetric marker for positive selection ofrecombinant raccoonpox virus is the E. coli uidA gene. Recombinantraccoonpox virus, S-RPV-011, was constructed to contain this DNAsequence.

FIG. 6: DNA sequence of the synthetic pox viral promoter LP2EP2 andposition of unique EcoRI and BamHI sites (SEQ ID NO. 29) for insertionof foreign DNA under the control of the LP2EP2 promoter in homologyvector 902-49.46, 902-67.1, 902-67.14, and 902-67.27. The colorimetricmarker for positive selection of recombinant raccoonpox virus is the E.coli lacZ gene. Recombinant raccoonpox viruses, S-RPV-007, S-RPV-008,S-RPV-009, S-RPV-010, was constructed to contain this DNA sequence indifferent insertion regions of the recombinant raccoonpox virus.

FIG. 7: DNA sequence of the raccoonpox virus E11L promoter and locationof the translation start codon. Direction of transcription is from rightto left. (SEQ ID NO. 30) The raccoonpox virus E11L promoter is usefulfor expression of foreign DNA in recombinant poxvirus vectors,including, but not limited to raccoonpox, swinepox, avipox, fowlpox,canarypox, cowpox, capripox, and vaccinia virus.

FIG. 8: Restriction endonuclease map of raccoonpox virus HindIII “S”genomic fragment (approximately 2,375 base pairs). At least two openreading frames (ORFs) are the B19R vaccinia virus homologue (Surfaceantigen; IL-1 receptor) (17) and the B18R variola virus homologue (787amino acids) (17); B20R vaccinia virus homologue (127 amino acids) (17).These three ORFs are non-essential for viral replication and suitablefor insertion of foreign DNA. Restriction endonuclease sites in the RPVHindIII S genomic fragment are shown as suitable insertion sites.Examples of recombinant raccoonpox of the present invention have foreignDNA inserted into the HpaI site at approximately nucleotide 4100(S-RPV-010) of the HindIII M fragment.

FIG. 9: DNA sequence of the swinepox virus I5L promoter and location ofthe translation start codon. Direction of transcription is from right toleft. (SEQ ID NO. 58) The swinepox virus I5L promoter is useful forexpression of foreign DNA in recombinant poxvirus vectors, including,but not limited to raccoonpox, swinepox, avipox, fowlpox, canarypox,cowpox, capripox, and vaccinia virus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a recombinant raccoonpox virus comprisinga raccoonpox virus viral genome which contains a foreign DNA sequenceinserted into a non-essential region within the HindIII “U” genomicregion of the raccoonpox region of the raccoonpox virus genome. Thepresent invention is useful for the expression of a foreign DNA or genesfrom other pathogens for protection against disease. Recombinantraccoonpox virus expressing a foreign DNA from avian or mammalianpathogens are useful as vaccines in avian or mammalian species includingbut not limited to feline, canine, equine, bovine, caprine, porcine, andprimate, including human.

As defined herein, “viral genome” means the entire DNA which naturallyoccurs in the virus. As defined herein, “foreign DNA” or “foreign gene”means any DNA or gene that is exogenous to a genome.

In one embodiment of the present invention, the foreign DNA sequence isinserted into an O1L open reading frame. In another embodiment, theforeign DNA is inserted into an E11 open reading frame. In anotherembodiment, the foreign DNA sequence is inserted into an intergenicregion. In another embodiment, the foreign DNA sequence is inserted intoan XbaI restriction endonuclease site.

A further embodiment of the recombinant raccoonpox virus comprising araccoonpox virus viral genome which contains a foreign DNA sequenceinserted into a non-essential region within the HindIII “U” genomicregion of the raccoonpox virus genome, further comprises the foreign DNAsequence inserted into the HindIII “U” genomic region, wherein theforeign DNA is capable of being expressed in a recombinant raccoonpoxvirus host cell.

One embodiment of the present invention is wherein the foreign DNAsequence encodes a screenable marker. Another embodiment is wherein thescreenable marker is E. coli beta-galactosidase or E. colibeta-glucuronidase. A further embodiment is wherein the foreign DNAsequence encodes an antigenic polypeptide. Another embodiment of thepresent invention is wherein the antigenic polypeptide when introducedinto the host cell, induces production of protective antibodies againsta feline disease-causing agent from which the antigenic polypeptide isderived or derivable. A further embodiment of the present invention iswherein the antigenic polypeptide is derived or derivable from a groupconsisting of feline pathogen, canine pathogen, equine pathogen, bovinepathogen, caprine pathogen, avian pathogen, porcine pathogen, or humanpathogen.

Another embodiment of the invention is wherein the foreign DNA sequenceis selected from a group consisting of swine influenza virushemagglutinin, swine influenza virus neuraminidase, porcine reproductiveand respiratory virus (PRRS) ORF2, PRRS ORF3, PRRS ORF4, PRRS ORF5, PRRSORF6, PRRS ORF7, porcine parvovirus VP2.

Another embodiment of the invention is wherein the foreign gene is undercontrol of an endogenous upstream, raccoonpox virus promoter. A furtherembodiment of the present invention is wherein promoter is selected froma group consisting of the raccoonpox virus E11L promoter and theraccoonpox virus O1L promoter. Another embodiment is wherein the foreigngene is under control of a heterologous upstream promoter.

A further embodiment of the invention is wherein the promoter isselected from a group consisting of the synthetic pox late promoter 1(LP1), the synthetic pox early promoter 1 (EP1), the synthetic earlypromoter 2 (EP2), the synthetic pox late promoter 2 early promoter 2(LP2EP2), the synthetic pox late promoter 1 (LP1), the synthetic poxlate promoter 2 (LP2), and the swinepox virus I5L promoter.

One embodiment of the invention is a recombinant raccoonpox virusdesignated S-RPV-001, designated S-RPV-002, designated S-RPV-003,designated S-RPV-004, designated S-RPV-005, designated S-RPV-006,designated S-RPV-007, designated S-RPV-011, designated S-RPV-012,designated S-RPV-013, designated S-RPV-015, designated S-RPV-016,designated S-RPV-017, designated S-RPV-018, designated S-RPV-019,designated S-RPV-020, or designated S-RPV-028.

The present invention also provides for a recombinant swinepox virus ofcomprising a swinepox virus genome which contains a foreign DNA sequenceinserted into a non-essential region of the swinepox virus genome,wherein the foreign DNA sequence is a host range gene selected from thegroup consisting of raccoonpox virus K1L and raccoonpox virus C7L. Oneembodiment of the present invention is the recombinant swinepox virusfurther comprising a second foreign DNA sequence inserted into anon-essential region within the swinepox virus genome. One embodiment ofthe present invention is the recombinant swinepox virus designatedS-SPV-248. Another embodiment of the present invention is therecombinant swinepox virus designated S-SPV-249.

The present invention also provides a recombinant raccoonpox viruscomprising a raccoonpox virus viral genome which contains a foreign DNAsequence inserted into a non-essential region within the HindIII “N”genomic region of the raccoonpox virus genome. A further embodiment ofthe present invention is wherein the foreign DNA sequence is insertedinto an SnaBI restriction endonuclease site. Another embodiment of thepresent invention is wherein the foreign DNA sequence is inserted intoan EcoRV restriction endonuclease site.

A preferred embodiment of the present invention is a recombinantraccoonpox virus designated S-RPV-008 or designated S-RPV-009.

The present invention provides for a recombinant raccoonpox viruscomprising a raccoonpox virus viral genome which contains a foreign DNAsequence inserted into a non-essential region within the HindIII “M”genomic region of the raccoonpox virus genome. One embodiment of thepresent invention is wherein the foreign DNA sequence is inserted intoan HpaI restriction endonuclease site. Another embodiment of the presentinvention is wherein the recombinant virus is designated S-RPV-010.

The present invention also provides for a recombinant raccoonpox viruscomprising a raccoonpox virus viral genome which contains a deletion ina raccoonpox virus host range gene of the viral genome, wherein theraccoonpox virus host range gene is selected from the group consistingof C7L, C6L, C5L, C4L, C3L, C2L, C1L, N1L, N2L, M1L, M2L, and K1L. Oneembodiment of the present invention is a recombinant raccoonpox virusfurther comprising a second foreign DNA sequence inserted into anon-essential region within the raccoonpox virus genome.

The present invention also provides for a homology vector for producinga recombinant raccoonpox virus by inserting a foreign DNA sequence intothe raccoonpox virus genome which comprises a double-stranded DNAmolecule consisting of: (a) double-stranded foreign DNA sequenceencoding an antigenic polypeptide derived from an animal pathogen; (b)at one end of the foreign DNA sequence, double-stranded feline virusgenomic DNA homologous to the genomic DNA located at one side of anon-essential site of the raccoonpox viral genomic DNA; (c) at the otherend of the foreign DNA sequence, double-stranded raccoonpox virusgenomic DNA homologous to the genomic DNA located at the other side ofthe same site.

In one embodiment of the homology vector the antigenic polypeptide isselected from a group consisting of swine influenza virus hemagglutinin,swine influenza virus neuraminidase, porcine reproductive andrespiratory virus (PRRS) ORF2, PRRS ORF3, PRRS, ORF4, PRRS ORF5, PRRSORF6, PRRS ORF7, porcine parvovirus VP2, E. coli beta-galactosidasegene., E. Coli beta-glucuronidase gene.

A further embodiment is a homology vector designated Homology Vector902-16.2. Another embodiment is a homology vector designated HomologyVector 902-19.18. A further embodiment is a homology vector designatedHomology Vector 902-49.5. A further embodiment is a homology vectordesignated Homology Vector 902-49.14. A further embodiment is a homologyvector designated Homology Vector 902-49.23.

Another embodiment is a homology vector designated Homology Vector902-49.34. A further embodiment is a homology vector designated HomologyVector 934-65.1, or Homology Vector 934-64.2 or Homology Vector 938-94.1or Homology Vector 938-94.25 or Homology Vector 950-13.4 or HomologyVector 936-87.2 or Homology Vector 950-16.34.

The present invention provides for a host cell infected with therecombinant raccoonpox virus comprising a raccoonpox virus viral genomewhich contains a foreign DNA sequence inserted into a non-essentialregion within the HindIII “U” genomic region of the raccoonpox virusgenome. One embodiment of the present invention is wherein the host cellis mammalian cell.

The present invention also provides for a vaccine against an animalpathogen which comprises an effective immunizing amount of therecombinant raccoonpox virus and a suitable carrier.

The present invention also provides for a method of immunizing an animalagainst an animal pathogen which comprises administering to the animalan effective immunizing dose of the vaccine.

The present invention also provides for a method of immunizing an animalagainst a feline pathogen which comprises administering to the animal aneffective immunizing dose of the vaccine.

The present invention provides for a recombinant raccoonpox viruscomprising a raccoonpox virus viral genome which contains a foreign DNAsequence inserted into a non-essential region within the HindIII “U”genomic region of the raccoonpox region of the raccoonpox virus genome.

In one embodiment the foreign DNA sequence is inserted into an O1L openreading frame.

In another embodiment the foreign DNA sequence is inserted into an E11Lopen reading frame.

In one embodiment the foreign DNA sequence is inserted into anintergenic region.

In a further embodiment the foreign DNA sequence is inserted into anXbaI restriction endonuclease site.

In another embodiment the recombinant raccoonpox virus offurthercomprises the foreign DNA sequence inserted into the HindIII “U” genomicregion, wherein the foreign DNA is capable of being expressed in arecombinant raccoonpox virus host cell.

In another embodiment the foreign DNA sequence encodes a screenablemarker.

In another embodiment the screenable marker is E. colibeta-galactosidase.

In another embodiment the screenable marker is E. colibeta-glucuronidase.

In one embodiment the foreign DNA sequence encodes an antigenicpolypeptide.

In another embodiment the antigenic polypeptide when introduced into thehost cell, induces production of protective antibodies against a felinedisease-causing agent from which the antigenic polypeptide is derived orderivable.

In another embodiment the antigenic polypeptide is derived or derivablefrom a group consisting of feline pathogen, canine pathogen, equinepathogen, bovine pathogen, caprine pathogen, avian pathogen, porcinepathogen, or human pathogen.

In another embodiment the foreign DNA sequence is selected from a groupconsisting of swine influenza virus hemagglutinin, swine influenza virusneuraminidase, porcine reproductive and respiratory virus (PRRS) ORF2,PRRS ORF3, PRRS ORF4, PRRS ORF5, PRRS ORF6, PRRS ORF7, porcineparvovirus VP2.

In another embodiment the foreign gene is under control of an endogenousupstream, raccoonpox virus promoter.

In another embodiment the promoter is selected from a group consistingof the raccoonpox virus E11L promoter and the raccoonpox virus O1Lpromoter.

In another embodiment the foreign gene is under control of aheterologous upstream promoter.

In another embodiment the promoter is selected from a group consistingof the synthetic pox late promoter 1 (LP1), the synthetic pox earlypromoter 1 (EP1), the synthetic pox early promoter 2 (EP2), thesynthetic pox late promoter 2 early promoter 2 (LP2EP2), the syntheticpox late promoter 1 (LP1), the synthetic pox late promoter 2 (LP2), andthe swinepox virus I5L promoter.

The present invention provides for a recombinant raccoonpox virusdesignated S-RPV-001, S-RPV-002, S-RPV-003 S-RPV-004, S-RPV-005,S-RPV-006, S-RPV-007, S-RPV-011, S-RPV-012, S-RPV-013, S-RPV-015,S-RPV-016, S-RPV-017, S-RPV-018, S-RPV-019, S-RPV-020, or S-RPV-028.

The present invention provides for a recombinant swinepox viruscomprising a swinepox virus genome which contains a foreign DNA sequenceinserted into a non-essential region of the raccoonpox virus genome,wherein the foreign DNA sequence is a host range gene selected from thegroup consisting of raccoonpox virus K1L and raccoonpox virus C7L.

In one embodiment the recombinant swinepox virus of further comprises asecond foreign DNA sequence inserted into a non-essential region withinthe swinepox virus genome.

In one embodiment the recombinant swinepox virus is designated S-SPV-248or S-SPV-249.

The present invention provides for a recombinant raccoonpox viruscomprising a raccoonpox virus viral genome which contains a foreignvirus viral genome which contains a foreign DNA sequence inserted into anon-essential region within the HindIII “N” genomic region of theraccoonpox virus genome.

In one embodiment the foreign DNA sequence is inserted into an EcoRIrestriction endonuclease site.

In another embodiment the foreign DNA sequence is inserted into an SnaBIrestriction endonuclease site.

The present invention provides for a recombinant raccoonpox virus ofdesignated S-RPV-008 or S-RPV-009.

The present invention provides for a recombinant raccoonpox viruscomprising a raccoonpox virus viral genome which contains a foreign DNAsequence inserted into a non-essential region within the HindIII “M”genomic region of the raccoonpox virus genome.

In one embodiment the foreign DNA sequence is inserted into an HpaIrestriction endonuclease site.

The present invention provides for a recombinant raccoonpox virusofdesignated S-RPV-010.

The present invetion provides for a recombinant raccoonpox viruscomprising a raccoonpox virus viral genome which contains a deletion ina raccoonpox virus host range gene of the viral genome, wherein theraccoonpox virus host range gene is selected from the group consistingof C7L, C6L, C5L, C4L, C3L, C2L, C1L, N1L, N2L, M1L, M2L, and K1L.

In one embodiment the recombinant raccoonpox virus of, further comprisesa second foreign DNA sequence inserted into a non-essential regionwithin the raccoonpox virus genome.

The present invention provides for a homology vector for producing arecombinant racconpox virus by inserting a foreign DNA sequence into theracconpox virus genome which comprises a double-stranded DNA moleculeconsisting of double-stranded foreign DNA sequence encoding an antigenicpolypeptide derived from an animal pathogen; at one end of the foreignDNA sequence, double-stranded feline virus genomic DNA homologous to thegenomic DNA located at one side of a non-essential site of theraccoonpox viral genomic DNA; at the other end of the foreign DNAsequence, double-stranded raccoonpox virus genomic DNA homologous to thegenomic DNA located at the other side of the same site.

In one embodiment the antigenic polypeptide is selected from a groupconsisting of swine influenza virus hemagglutinin, swine influenza virusneuraminidase, porcine reproductive and respiratory virus (PRRS) ORF2,PRRS ORF3, PRRS, ORF4, PRRS ORF5, PRRS ORF6, PRRS ORF7, porcineparvovirus VP2, E. coli beta-galactosidase gene., and E. Colibeta-glucuronidase gene.

The present invention provides for a homology vector designated HomologyVector 902-16.2, Homology Vector 902-19.18, Homology Vector 902-49.5,Homology Vector 902-49.14, Homology Vector 902-49.23, Homology Vector902-49.34, Homology Vector 934.65-1, Homology Vector 934-64.2, HomologyVector 938-94.1 Homology Vector 938-94.25, Homology Vector 950-13.4,Homology Vector 936-87.2, Homology Vector 950-16.34,

The present invention provides for a host cell infected with arecombinant raccoonpox comprising a raccoonpox virus viral genome whichcontains a foreign DNA sequence inserted into a non-essential regionwithin the HindIII “U” genomic region of the raccoonpox region of theraccoonpox virus genome.

In one embodiment the host cell is mammalian cell.

The present invention provides for a vaccine against an animal pathogenwhich comprises an effective immunizing amount of a recombinantraccoonpox virus and a suitable carrier.

The present invention provides for a method of immunizing an animalagainst an animal pathogen which comprises administering to the animalan effective immunizing dose of a recombinant raccoonpox virus and asuitable carrier.

The present invention provides for a method of immunizing an animalagainst a feline pathogen which comprises administering to the animal aneffective immunizing dose of a recombinant raccoonpox virus and asuitable carrier.

The invention provides a recombinant raccoonpox virus capable ofreplication which contains foreign DNA encoding an antigenic polypeptidewhich is useful to prevent disease in porcine, bovine, feline, canine,avian, equine, caprine and human species.

The invention provides a recombinant raccoonpox virus capable ofreplication which contains foreign DNA encoding an antigenic polypeptidewhich is or is from pseudorabies virus (PRV) g50 (gD), pseudorabiesvirus (PRV) gII (gB), Pseudorabies virus (PRV) gIII (gC), pseudorabiesvirus (PRV) glycoprotein H, pseudorabies virus (PRV) glycoprotein E,Transmissible fungastroenteritis (TGE) glycoprotein 195, Transmissiblegastroenteritis (TGE) matrix protein, swine rotavirus glycoprotein 38,swine parvovirus capsid protein, Serpulina hydodysenteriae protectiveantigen, Bovine Viral Diarrhea (BVD) glycoprotein 55, swine influenzahemagglutinin or swine influenza neuraminidase. Preferably, theantigenic polypeptide is Pseudorabies Virus (PRV) g50 (gD). Preferablythe antigenic polypeptide is swine influenza hemagglutinin or swineinfluenza neuraminidase.

The present invention further provides an antigenic polypeptide whichincludes, but is not limited to: hog cholera virus gEl, hog choleravirus gE2, swine influenza virus hemagglutinin, neurominidase, matrixand nucleoprotein, pseudorabies virus gB, gC and gD, and porcinereproductive and respiratory disease (PRRS) virus ORF2, ORF3, ORF4,ORF5, ORF6 and ORF7. Preferably the antigenic polypeptide is PRRS, ORF2,ORF3, ORF4, ORF5, ORF6, and ORF 7.

The invention further provides a recombinant raccoonpox virus capable ofreplication which contains foreign DNA encoding an antigenic polypeptidewhich is or is from Serpulina hyodysenteriae, Foot and Mouth DiseaseVirus, Hog Cholera Virus, Swine Influenza Virus, African Swine FeverVirus or Mycoplasma hyopneumoniae.

The invention further provides a recombinant raccoonpox virus capable ofreplication which contains foreign DNA encoding an antigenic polypeptidewhich is or is from feline leukemia virus, feline immunodeficiency virusand Dirofilaria immitis (heartworm). Additional antigens from diseasecausing microorganisms are expressed in raccoonpox virus. Diseasecausing microorganisms in cats include, but are not limited toDirofilaria immitis p39 and 22kD antigens, feline infectious peritonitisvirus, calicivirus, rabies virus, feline parvovirus (panleukopeniavirus), feline coronavirus and feline Chlamydia, Toxoplasma gondii.Disease causing microorganisms in dogs include, but are not limited tocanine distemper, canine adenovirus type 1 (hepatitis), adenovirus type2 (respiratory disease), parainfluenza, leptospira canicola,icterohemorragia, parvovirus, coronavirus, borrelia burgdorferi, canineherpesvirus, bordetella bronchiseptica and rabies virus.

The present invention further provides a recombinant raccoonpox viruswhich comprises a foreign DNA sequence inserted into a non-essentialsite of the raccoonpox genome, wherein the foreign DNA sequence encodesan antigenic polypeptide derived from infectious laryngotracheitis virusand is capable of being expressed in a host infected by the recombinantraccoonpox virus. Examples of such antigenic polypeptide are infectiouslaryngotracheitis virus glycoprotein G, glycoprotein D and glycoproteinI, Newcastle Disease Virus (NDV) hemagglutinin-neuraminidase, NDV fusionprotein (F).

In one embodiment of the recombinant raccoonpox virus the foreign DNAsequence encodes a cytokine. In another embodiment the cytokine ischicken myelomonocytic growth factor (cMGF) or chicken interferon(cIFN). Cytokines include, but are not limited to: transforming growthfactor beta, epidermal growth factor family, fibroblast growth factors,hepatocyte growth factor, insulin-like growth factor, vascularendothelial growth factor, interleukin 1, IL-1 receptor antagonist,interleukin-2, interleukin-3, interleukin-4, interleukin-5,interleukin-6, IL-6 soluble receptor, interleukin-7, interleukin-8,interleukin-9, interleukin-10, interleukin-11, interleukin-12,interleukin-13, interleukin-14, interleukin-15, interleukin-16,angiogenin, chemokines, colony stimulating factors,granulocyte-macrophage colony stimulating factors, erythropoietin,interferon, interferon gamma, c-kit ligand, leukemia inhibitory factor,oncostatin M, pleiotrophin, secretory leukocyte protease inhibitor, stemcell factor, tumor necrosis factors, and soluble TNF receptors. Thesecytokines are from humans, bovine, equine, feline, canine, porcine oravian.

The present invention further provides a recombinant raccoonpox viruswhich comprises a foreign DNA sequence inserted into a non-essentialsite of the raccoonpox genome, wherein the foreign DNA sequence encodesan antigenic polypeptide derived from a human pathogen and is capable ofbeing expressed in a host infected by the recombinant raccoonpox virus.

Recombinant RPV expressing cytokines is used to enhance the immuneresponse either alone or when combined with vaccines containingcytokines or antigen genes of disease causing microorganisms.

Antigenic polypeptide of a human pathogen which are derived from humanherpesvirus include, but are not limited to: hepatitis B virus andhepatitis C virus hepatitis B virus surface and core antigens, hepatitisC virus, human immunodeficiency virus, herpes simplex virus-1, herpessimplex virus-2, human cytomegalovirus, Epstein-Barr virus,Varicella-Zoster virus, human herpesvirus-6, human herpesvirus-7, humaninfluenza, measles virus, hantaan virus, pneumonia virus, rhinovirus,poliovirus, human respiratory syncytial virus, retrovirus, human T-cellleukemia virus, rabies virus, mumps virus, malaria (Plasmodiumfalciparum), Bordetella pertussis, Diphtheria, Rickettsia prowazekii,Borrelia burgdorferi, Tetanus toxoid, malignant tumor antigens.

The present invention further provides a recombinant raccoonpox viruswhich comprises a foreign DNA sequence inserted into a non-essentialsite of the raccoonpox genome, wherein the foreign DNA sequence encodesa cytokine capable of stimulating an immune in a host infected by therecombinant raccoonpox virus and is capable of being expressed in thehost infected.

The antigenic polypeptide of an equine pathogen can derived from equineinfluenza virus, or equine herpesvirus. In one embodiment the antigenicpolypeptide is equine influenza neuraminidase or hemagglutinin. Examplesof such antigenic polypeptide are equine influenza virus type A/Alaska91 neuraminidase, equine influenza virus type A/Prague 56 neuraminidase,equine influenza virus type A/Miami 63 neuraminidase, equine influenzavirus type A/Kentucky 81 neuraminidase, equine influenza virus typeA/Kentucky 92 neuraminidase equine herpesvirus type 1 glycoprotein B,equine herpesvirus type 1 glycoprotein D, Streptococcus equi, equineinfectious anemia virus, equine encephalitis virus, equine rhinovirusand equine rotavirus.

The present invention further provides a recombinant raccoonpox viruswhich comprises a foreign DNA sequence inserted into a non-essentialsite of the raccoonpox genome, wherein the foreign DNA sequence encodesan antigenic polypeptide derived from bovine respiratory syncytial virusor bovine parainfluenza virus, and is capable of being expressed in ahost infected by the recombinant raccoonpox virus.

For example, the antigenic polypeptide of derived from infectious bovinerhinotracheitis virus gE, bovine respiratory syncytial virus equinepathogen can derived from equine influenza virus is bovine respiratorysyncytial virus attachment protein (BRSV G), bovine respiratorysyncytial virus fusion protein (BRSV F), bovine respiratory syncytialvirus nucleocapsid protein (BRSV N), bovine parainfluenza virus type 3fusion protein, and the bovine parainfluenza virus type 3 hemagglutininneuraminidase.

The present invention further provides a recombinant raccoonpox viruswhich comprises a foreign DNA sequence inserted into a non-essentialsite of the raccoonpox genome, wherein the foreign DNA sequence encodesan antigenic polypeptide derived from infectious bovine rhinotracheitisvirus and is capable of being expressed in a host infected by therecombinant raccoonpox virus. Examples of such antigenic polypeptide areinfectious bovine rhinotracheitis virus glycoprotein E, glycoprotein G,glycoprotein gD and glycoprotein gI.

The present invention further provides a recombinant raccoonpox viruswhich comprises a foreign DNA sequence inserted into a non-essentialsite of the raccoonpox genome, wherein the foreign DNA sequence encodesbovine viral diarrhea virus (BVDV) glycoprotein 48 or glycoprotein 53,and wherein the foreign DNA sequence is capable of being expressed in ahost infected by the recombinant raccoonpox virus.

The present invention further provides a recombinant raccoonpox viruswhich comprises a foreign DNA sequence inserted into a non-essentialsite of the raccoonpox genome, wherein the foreign DNA sequence encodesan antigenic polypeptide derived from infectious bursal disease virusand wherein the foreign DNA sequence is capable of being expressed in ahost infected by the recombinant raccoonpox virus. Examples of suchantigenic polypeptide are infectious bursal disease virus polyproteinand VP2.

The present invention further provides a recombinant raccoonpox virus inwhich the foreign DNA sequence encodes an antigenic polypeptide whichincludes, but is not limited to: MDV gA, MDV gB, MDV gD, NDV HN, NDV F,ILT gB, ILT gI, ILT gD, IBDV VP2, IBDV VP3, IBDV VP4, IBDV polyprotein,IBV spike, IBV matrix, avian encephalomyelitis virus, avian reovirus,avian paramyxovirus, avian influenza virus, avian adenovirus, fowl poxvirus, avian coronavirus, avian rotavirus, chick anemia virus,Salmonella spp. E. coli, Pasteurella spp., Bordetella spp., Eimeriaspp., Histomonas spp., Trichomonas spp., Poultry nematodes, cestodes,trematodes, poultry mites/lice, and poultry protozoa.

This invention is illustrated in the Experimental Detail sections whichfollow. These sections are set forth to aid in an understanding of theinvention but are not intended to, and should not be construed to, limitin any way the invention as set forth in the claims which followthereafter.

EXPERIMENTAL DETAILS

Preparation of Raccoonpox Virus Stock Samples

Raccoonpox virus (RPV) isolate ATCC VR-838 was used for preparation ofraccoonpox virus stock samples and raccoonpox virus genomic DNA. AnotherRPV isolate available is V71-I-85A from Center for Disease Control (CDC;Atlanta, Ga.). Raccoonpox virus (RPV) samples were prepared by infectingVERO cells, CRFK cells or MDCK cells at a multiplicity of infection of0.01 PFU/cell in Dulbeccols Modified Eagle's Medium containing 2 mMglutamine, 100 units/ml penicillin, 100 units/ml streptomycin (thesecomponents were obtained from Sigma or equivalent supplier, andhereafter are referred to as DMEM negative medium). Prior to infection,the cell monolayers were washed once with DMEM negative medium to removetraces of fetal bovine serum. The RPV contained in the initial inoculum(0.5 ml for 10 cm plate; 10 ml for T225 cm flask) was then allowed toabsorb onto the cell monolayer for two hours, being redistributed everyhalf hour. After this period, the original inoculum was brought up tothe recommended volume with the addition of complete DMEM medium (DMEMnegative medium plus 5% fetal bovine serum). The plates were incubatedat 37° C. in 5% CO₂ until cytopathic effect was complete. The medium andcells were harvested and frozen in a 50 ml conical screw cap tube at−70° C. Upon thawing at 37° C., the virus stock was aliquoted into 1.0ml vials and refrozen at −70° C. The titers were usually about 10⁵-10⁶PFU/ml.

Preparation of RPV DNA

For raccoonpox virus DNA isolation, a confluent monolayer of VERO cellsin a T225 cm² flask was infected at a multiplicity of 0.1 withraccoonpox virus (ATCC VR-838) and incubated 3-5 days until the cellswere showing 100% cytopathic effect. The infected cells were thenharvested by scraping the cells into the medium and centrifuging at 3000rpm for 5 minutes in a clinical centrifuge. The medium was decanted, andthe cell pellet was gently resuspended in 1.0 ml Phosphate Buffer Saline(PBS: 1.5 g Na₂HPO₄, 0.2 g KH₂PO_(4,) 0.8 g NaCl and 0.2 g KCl per literH₂O) (per T175) and subjected to two successive freeze-thaws (−70° C. to37° C.). Upon the last thaw, the cells (on ice) were sonicated two timesfor 30 seconds each with 45 seconds cooling time in between. Cellulardebris was then removed by centrifuging (Sorvall RC-5B superspeedcentrifuge) at 3000 rpm for 5 minutes in a HB4 rotor at 4° C. RPVvirions, present in the supernatant, were then pelleted bycentrifugation at 15,000 rpm for 20 minutes at 4° C. in a SS34 rotor(Sorvall) and resuspended in 10 mM Tris (pH 7.5). This fraction was thenlayered onto a 36% sucrose gradient (w/v in 10 mM tris pH 7.5) andcentrifuged (Beckman L8-70M Ultracentrifuge) at 18,000 rpm for 60minutes in a SW41 rotor (Beckman) at 4° C. The virion pellet wasresuspended in 1.0 ml of 10 mM tris pH 7.5 and sonicated on ice for 30seconds. This fraction was layered onto a 20% to 50% continuous sucrosegradient and centrifuged 16,000 rpm for 60 minutes in a SW41 rotor at 4°C. The RPV virion band located about three quarters down the gradientwas harvested, diluted with 20% sucrose and pelleted by centrifugationat 18,000 rpm for 60 minutes in a SW41 rotor at 4° C. The resultantpellet was then washed once with 10 mM Tris pH 7.5 to remove traces ofsucrose and finally resuspended in 10 mM Tris pH 7.5. RPV DNA was thenextracted from the purified virions by lysis (4 hours at 60° C.) inducedby the addition of EDTA, SDS, and proteinase K to final concentrationsof 20 mM, 0.5% and 0.5 mg/ml, respectively. After digestion, threephenol:chloroform (1:1) extractions were conducted and the sampleprecipitated by the addition of two volumes of absolute ethanol andincubation at −20° C. for 30 minutes. The sample was then centrifuged inan Eppendorf minifuge for 5 minutes at full speed. The supernatant wasdecanted, and the pellet air dried and rehydrated in 0.01 M Tris pH 7.5,1 mM EDTA at 4° C.

Preparation of Infected Cell Lysates

For cell lysate preparation, serum free medium was used. A confluentmonolayer of cells (VERO, CRFK, or MDCK) in a 25 cm flask or a 60 mmpetri dish was infected with 100 μl of virus sample. After cytopathiceffect was complete, the medium and cells were harvested and the cellswere pelleted at 3000 rpm for 5 minutes in a clinical centrifuge. Thecell pellet was resuspended in 250 μl of disruption buffer (2% sodiumdodecyl sulfate, 2% β-mercapto-ethanol). The samples were sonicated for30 seconds on ice and stored at −20° C.

Western Blotting Procedure

Samples of lysates and protein standards were run on a polyacrylamidegel according to the procedure of Laemnli (19). After gelelectrophoresis the proteins were transferred and processed according toSambrook et al. (1989) (20). The primary antibody was diluted 1:100 with5% non-fat dry milk in Tris-sodium chloride, and sodium Azide (TSA: 6.61g Tris-HCl, 0.97 g Tris-base, 9.0 g NaCl and 2.0 g Sodium Azide perliter H₂O). The secondary antibody was alkaline phosphatase conjugatedand diluted 1:1000 with TSA.

Molecular Biological Techniques

Techniques for the manipulation of bacteria and DNA, including suchprocedures as digestion with restriction endonucleases, gelelectrophoresis, extraction of DNA from gels, ligation, phosphorylationwith kinase, treatment with phosphatase, growth of bacterial cultures,transformation of bacteria with DNA, and other molecular biologicalmethods are described by Sambrook et al. (1989) and Current Protocols inMolecular Biology (1992). (20,21) Except as noted, these were used withminor variation.

DNA Sequencing

DNA sequencing was performed by fluorescent labeled dideoxy sequencingreactions using ABI PRISM Dye Terminator Cycle Sequencing Ready ReactionKit with AMPLITAQ DNA polymerase, FS (Perkin-Elmer; per manufacturer'sinstructions) and electrophoresed on an Perkin-Elmer/Applied Biosystemsautomated DNA sequencer Model 373A according to manufacturer'sinstructions. Reactions using both the dGTP mixes and the dITP mixeswere performed to clarify areas of compression.

Alternatively, compressed areas were resolved on formamide gels.Templates were double-stranded plasmid subclones or single stranded M13subclones, and primers were either made to the vector just outside theinsert to be sequenced, or to previously obtained sequence. Sequenceobtained was assembled and compared using DNAStar software.

Cloning with the Polymerase Chain Reaction

The polymerase chain reaction (PCR) was used to introduce restrictionsites convenient for the manipulation of various DNAs. The proceduresused are described by Innis, et al. (1990) (22). In general, amplifiedfragments were less than 500 base pairs in size and critical regions ofamplified fragments were confirmed by DNA sequencing. The primers usedin each example are detailed in the descriptions of the construction ofhomology vectors below.

Homologous Recombination Procedure for Generating Recombinant RPV or SPV

This method relies upon the homologous recombination between theraccoonpox virus DNA and the plasmid homology vector DNA which occurs inthe tissue culture cells containing both raccoonpox virus DNA orswinepox virus DNA and transfected plasmid homology vector. Forhomologous recombination to occur, the monolayers of cells (CRFK, MDCK,ESK-4, PK15 or VERO) are infected with S-RPV-000 (ATCC VR-838) orS-SPV-001 at a multiplicity of infection of 0.01 PFU/cell to introducereplicating RPV or SPV (i.e. DNA synthesis) into the cells. The plasmidhomology vector DNA is then transfected into these cells according tothe INFECTION—TRANSFECTION PROCEDURE. The construction of homologyvectors used in this example of the procedure is described below.

Infection—transfection Procedure for RPV or SPV

6 cm plates of cells (CRFK, MDCK, ESK4, or VERO) about 80% confluentwere infected with S-RPV-000 or SPV-001 at a multiplicity of infectionof 0.01 PFU/cell in DMEM negative medium or ESK negative medium andincubated at 37° C. in a humidified 5% CO₂ environment for 2-3 hours.The transfection procedure used is essentially that recommended forLIPOFECTIN Reagent (BRL). Briefly, for each 6 cm plate, 15 μg of plasmidDNA was diluted up to 100 μl with H₂O. Separately, 50 micrograms ofLIPOFECTIN™ Reagent was diluted to 100 μl with H₂O. The 100 μl ofdiluted LIPOFECTIN™ Reagent was then added dropwise to the dilutedplasmid DNA contained in a polystyrene 5 ml snap cap tube and mixedgently. The mixture was then incubated for 15-20 minutes at roomtemperature. During this time, the virus inoculum was removed from the 6cm plates and the cell monolayers washed once with either DMEM or ESKnegative medium. Five ml of DMEM negative medium or ESK negative mediumwas then added to the plasmid DNA/LIPOFECTIN™ mixture and the contentspipetted onto the cell monolayer. The cells were incubated overnight(about 16 hours) at 37° C. in a humidified 5% CO₂ environment. The nextday the 5 ml of DMEM negative medium was removed and replaced with 5 mlDMEM complete medium. The cells were incubated at 37° C. in 5% CO₂ for3-5 days until cytopathic effect from the virus was 80-100%. Virus washarvested as described above for the preparation of virus stocks. Thisstock was referred to as a transfection stock and was subsequentlyscreened for recombinant virus by the BLUOGAL™ SCREEN FOR RECOMBINANTRACCOONPOX VIRUS OR CPRG SCREEN FOR RECOMBINANT RACCOONPOX VIRUS.

Screen for Recombinant RPV or SPV Expressing β-galactosidase BLUOGAL™and CPRG assays) or β-glucuronidase (X-GLUC™ assay)

When the E. coli β-galactosidase (lacZ) marker gene was incorporatedinto a recombinant virus the plaques containing the recombinants werevisualized by one of two simple methods. In the first method, thechemical BLUOGAL™ (Life Sciences Technology, Bethesda, Md.) wasincorporated (200 μg/ml) into the agarose overlay during the plaqueassay, and plaques expressing active β-galactosidase turned blue. Theblue plaques were then picked onto fresh cells (MDCK, ESK4, CRFK orVERO) and purified by further blue plaque isolation. In the secondmethod, CPRG (Boehringer Mannheim) was incorporated (400 μg/ml) into theagarose overlay during the plaque assay, and plaques expressing activeβ-galactosidase turned red. The red plaques were then picked onto freshcells (MDCK, ESK4 CRFK or VERO) and purified by further red plaqueisolation. In both cases viruses were typically purified with three tofour rounds of plaque purification.

When the E. coli β-glucuronidase (uidA) marker gene was incorporatedinto a recombinant virus the plaques containing the recombinants werevisualized by using the chromogenic substrate, X-beta-D-gluUA CHX(X-GLUC™; 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid,cyclohexylammonium salt; Biosynth AG; Switzerland) was incorporated (200μg/ml) into the agarose overlay during the plaque assay, and plaquesexpressing active β-glucuronidase turned blue. The blue plaques werethen picked onto fresh cells (MDCK, ESK4 CRFK or VERO) and purified byfurther blue plaque isolation.

Screen for Foreign Gene Expression in Recombinant RPV using Black PlaqueAssays

To analyze expression of foreign antigens expressed by recombinantraccoonpox viruses, monolayers of cells (MDCK, CRFK or VERO) wereinfected with recombinant RPV, overlayed with nutrient agarose media andincubated for 3-5 days at 37° C. for plaque development to occur. Theagarose overlay was then removed from the dish, the cells fixed with100% methanol for 10 minutes at room temperature and the cells airdried. Fixation of the cells results in cytoplasmic antigen as well assurface antigen detection whereas specific surface antigen expressioncan be detected using non-fixed cells. The primary antibody was thendiluted to the appropriate dilution with 1×blotto (5% non-fat dry milkin Tris-sodium chloride, and sodium Azide (TSA: 6.61 g Tris-HCl, 0.97 gTris-base, 9.0 g NaCl and 2.0 g Sodium Azide per liter H₂O) andincubated on the cell monolayer for 2 hours at room temperature. Unboundantibody was then removed by washing the cells three times with TSbuffer at room temperature. The secondary antibody, aalkaline-phosphatase conjugate, was diluted 1:1000 with 1×blotto andincubated with the cells for 2 hours at room temperature. Unboundsecondary antibody was then removed by washing the cells three timeswith TS buffer (6.61 g Tris-HCl, 0.97 g Tris-base, 9.0 g NaCl per literH₂O) at room temperature. The cells were then incubated 15-30 minutes atroom temperature with freshly prepared substrate solution (100 mM TrisHCl pH. 9.5, 100 mM NaCl, 5 mM MgCl₂, 0.3 mg/ml Nitro Blue Tetrazoliumand 0.15 mg/ml 5-Bromo-4-chloro-3-Indoyl Phosphatas). Plaques expressingthe correct antigen stain black. A fixer solution (20 mM Tris-HCl pH 2.9and 1 mM EDTA) was used to stop the color development reaction.

Procedure for Purification of Viral Glycoproteins for use as Diagnostics

Viral glycoproteins are purified using antibody affinity columns. Toproduce monoclonal antibodies, 8 to 10 week old BALB/c female mice arevaccinated intraperitoneally seven times at two to four week intervalswith 10⁷ PFU of raccoonpox virus recombinants. Three weeks after thelast vaccination, mice are injected intraperitoneally with 40 mg of thecorresponding viral glycoprotein. Spleens are removed from the micethree days after the last antigen dose.

Splenocytes are fused with mouse NS1/Ag4 plasmacytoma cells by theprocedure modified from Oi and Herzenberg, (23). Splenocytes andplasmacytoma cells are pelleted together by centrifugation at 300×g for10 minutes. One ml of a 50% solution of polyethylene glycol (m.w.1300-1600) is added to the cell pellet with stirring over one minute.Dulbecco's modified Eagles's medium (5 ml) is added to the cells overthree minutes. Cells are pelleted by centrifugation at 300×g for 10minutes and resuspended in medium with 10% fetal bovine serum andcontaining 100 mM hypoxanthine, 0.4 mM aminopterin and 16 mM thymidine(HAT). Cells (100 ml) are added to the wells of eight to ten 96-welltissue culture plates containing 100 ml of normal spleen feeder layercells and incubated at 37° C. Cells are fed with fresh HAT medium everythree to four days.

Hybridoma culture supernatants are tested by the ELISA ASSAY in 96-wellmicrotiter plates coated with 100 ng of viral glycoprotein. Supernatantsfrom reactive hybridomas are further analyzed by black-plaque assay andby Western Blot. Selected hybridomas are cloned twice by limitingdilution. Ascetic fluid is produced by intraperitoneal injection of5×10⁶ hybridoma cells into pristane-treated BALB/c mice.

Cell lysates from raccoonpox virus recombinants are obtained asdescribed in PREPARATION OF INFECTED CELL LYSATES. Theglycoprotein-containing cell lysates (100 mls) are passed through a 2-mlagarose affinity resin to which 20 mg of glycoprotein monoclonalantibody has been immobilized according to manufacturer's instructions(AFC Medium, New Brunswick Scientific, Edison, N.J.). The column iswashed with 100 ml of 0.1% Nonidet P-40 in phosphate-buffered saline(PBS) to remove nonspecifically bound material. Bound glycoprotein iseluted with 100 mM carbonate buffer, pH 10.6 (24). Pre- and postelutedfractions are monitored for purity by reactivity to the RPV monoclonalantibodies in an ELISA system.

Elisa Assay

A standard enzyme-linked immunosorbent assay (ELISA) protocol is used todetermine the immune status of cattle following vaccination andchallenge.

A glycoprotein antigen solution (100 ml at ng/ml in PBS) is allowed toabsorb to the wells of microtiter dishes for 18 hours at 4° C. Thecoated wells are rinsed one time with PBS. Wells are blocked by adding250 ml of PBS containing 1% BSA (Sigma) and incubating 1 hour at 37° C.The blocked wells are rinsed one time with PBS containing 0.02% Tween20. 50 ml of test serum (previously diluted 1:2 in PBS containing 1%BSA) are added to the wells and incubated 1 hour at 37° C. The antiserumis removed and the wells are washed 3 times with PBS containing 0.02%Tween 20. 50 ml of a solution containing anti-bovine IgG coupled tohorseradish peroxidase (diluted 1:500 in PBS containing 1% BSA,Kirkegaard and Perry Laboratories, Inc.) is added to visualize the wellscontaining antibody against the specific antigen. The solution isincubated 1 hour at 37° C., then removed and the wells are washed 3times with PBS containing 0.02% Tween 20. 100 ml of substrate solution(ATBS, Kirkegaard and Perry Laboratories, Inc.) are added to each welland color is allowed to develop for 15 minutes. The reaction isterminated by addition of 0.1M oxalic acid. The color is read atabsorbance 410 nm on an automatic plate reader.

Strategy for the Construction of Synthetic Pox Viral Promoters

For recombinant swinepox vectors synthetic pox promoters offer severaladvantages including the ability to control the strength and timing offoreign gene expression. Three promoter cassettes LP1, EP1 and LP2 basedon promoters that have been defined in the vaccinia virus (1, 7 and 8)were designed. Each cassette was designed to contain the DNA sequencesdefined in vaccinia flanked by restriction sites which could be used tocombine the cassettes in any order or combination. Initiator methionineswere also designed into each cassette such that inframe fusions could bemade at either EcoRI or BamHI sites. A set of translational stop codonsin all three reading frames and an early transcriptional terminationsignal were also engineered downstream of the inframe fusion site. DNAencoding each cassette was synthesized according to standard techniquesand cloned into the appropriate homology vectors (see FIGS. 5, 6, and7).

Procedure for Production of Murine Polyclonal Antibodies to ProteinsExpressed in Recombinant Raccoonpox Virus

Three groups of five mice each were vaccinated subcutaneously in therear leg with 200 ul of a 1:1 mixture of infected cell lysate andFreunds complete adjuvant (S-RPV-003 (SIV NA), S-RPV-004 (PRRS ORF3) andS-RPV-005 (PRRS ORF4)). The infected cell lysates were prepared byharvesting a T225 flask Vero cells at 90%+CPE. The infected cell pelletswere washed once in PBS and finally resuspended in 1 ml of PBS. The 1 mllysate was frozen at −70° C. and thawed on ice. The 1 ml lysate wassonicated one time (lowest setting). The mice received two additionaldoses of cell lysates mixed with Freunds incomplete adjuvant at 3 weekspost vaccination and 6 weeks post vaccination. The mice were sacrificedat 8 weeks post vaccination. About 2 ml of serum was obtained from 5mice. The serum was tested for SIV or PRRS specific antibodies by BLACKPLAQUE ASSAY.

Plasmid 880-96.43

The plasmid 880-96.43 was constructed for the purpose of insertingforeign DNA into raccoonpox virus. It comprises the approximately 2055base pair HindIII U genomic fragment of raccoonpox virus (SEQ ID NO. 1).Two open reading frames within the HindIII U fragment are the vacciniavirus homologs (17) of the O1L ORF (SEQ ID NO. 2) and the E11L ORF (SEQID NO. 3). The O1L ORF and E11L ORF are non-essential and foreign DNA isinserted within these ORFs or in the intergenic region between the ORFs.Any restriction site within this region is useful as an insertion sitefor foreign DNA. A restriction enzyme site within this region which isnot unique is altered by insertion of a DNA linker which converts thesite to a unique restriction enzyme recognition sequence. Preferably therestriction enzyme site used for insertion of foreign DNA is an XbaIsite at approximately nucleotide 1065 within the 2055 base pair HindIIIU genomic fragment (SEQ ID NO. 1). The insertion site is within the O1LORF between amino acids 459 and 460 of the open reading frame. Theplasmid vector was derived from an approximately 2999 base pair HindIIIrestriction fragment of pSP64 (PROMEGA™). Fragment 1 is an approximately2055 base pair HindIII U genomic fragment of raccoonpox virus (6) (SEQID NO. 1). Plasmid 880-96.43 was used to make homology vectors forinsertion of foreign DNA in recombinant raccoonpox virus.

Plasmid 902-15.2

The plasmid 902-15.2 was constructed for the purpose of insertingforeign DNA into raccoonpox virus. It comprises the approximately 1801base pair subfragment (SEQ ID NO. 4) of the HindIII U genomic fragmentof raccoonpox virus. Two open reading frames within the HindIII Ufragment are the vaccinia virus homologs (17) of the O1L ORF (SEQ ID NO.2) and the E11L ORF (SEQ ID NO. 3). The O1L ORF and E11L ORF arenon-essential and foreign DNA is inserted within these ORFs or in theintergenic region between the ORFs. Any restriction site within thisregion is useful as an insertion site for foreign DNA. The plasmidvector was derived from an approximately 2999 base pair HindIIIrestriction fragment of pSP64 (PROMEGA™). Fragment 1 is an approximately1801 base pair HindIII subfragment of the HindIII U genomic fragment (6)synthesized by POLYMERASE CHAIN REACTION using plasmid 880-96.43 as atemplate. The primer upstream of the O1L and E1L ORFs was5-CCGAAGCTTCCGTGCTCCATCTATATAATATATTAAAC-3′ (SEQ ID NO 14; 2/97.6). Theprimer downstream of the O1L and E11L ORFs was5′-GAATAAGCTTCCCGTTACTTTAGTAAAATCTTTTACAAA-3′ (SEQ ID NO 15; 2/97.7). Aunique NotI synthetic linker was inserted within an XbaI site(Nucleotides 1098 to 1113; a unique NotI site is at nucleotide 1105; SEQID NO. 4)

The restriction enzyme site used for insertion of foreign DNA is an XbaIsite at approximately nucleotide 1065 within the 2055 base pair HindIIIU genomic fragment (SEQ ID NO. 1). The insertion site is within the O1LORF between amino acids 459 and 460 of the open reading frame. Plasmid902-15.2 was used to make homology vectors for insertion of foreign DNAin recombinant raccoonpox virus.

Homology Vector 902-16.2

The plasmid 902-16.2 was constructed for the purpose of insertingforeign DNA into recombinant raccoonpox virus (RPV). It incorporates anE. coli β-galactosidase (lacZ) marker gene and a swine influenza virus(SIV) HA (H1N1) gene flanked by RPV DNA. Upstream of the foreign gene isan approximately 906 base pair fragment of RPV DNA. Downstream of theforeign genes is an approximately 895 base pair fragment of RPV DNA.Direction of transcription of the SIV HA gene is opposite the directionof transcription of the lacZ gene and opposite to the direction oftranscription of the RPV O1L ORF. When the plasmid is used according tothe HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT RPV, avirus containing DNA coding for the foreign genes will result. Note thatthe 3-galactosidase (lacZ) marker gene is under the control of a latepromoter (LP1) and the SIV HA (H1N1) gene is under the control of thelate/early promoter (LP2EP2). It was constructed utilizing standardrecombinant DNA techniques (20, 21), by joining restriction fragmentsfrom the following sources with the synthetic DNA sequences. The plasmidvector was derived from an approximately 2999 base pair HindIIIrestriction fragment of pSP64 (PRONEGA™). Fragment 1 is an approximately906 base pair HindIII to XbaI restriction sub-fragment of the RPVHindIII restriction fragment U (6). Fragment 2 is an approximately 1721base pair BamHI to BamHI restriction fragment synthesized by reversetranscription and polymerase chain reaction (PCR) using genomic RNA fromthe SIV H1N1 strain (NVSL) To synthesize SIV HA (H1N1, the primer5′-CCGAGGATCCGGCAATACTATTAGTCTTGCTATGTACATT-3′; 6/95.5) (SEQ ID NO 16)synthesized from the 5′ end of the SIV HA (H1N1) gene, introduced anBamHI site at the 5′ end of the gene. The primer(5′-CTCTGGGATCCTAATTTTAAATACATATTCTGCACTGTA-3′; 6/95.6) (SEQ ID NO 17)was used for reverse transcription and PCR and synthesized from the 3′end of the SIV HA (H1N1) gene and introduced a BamHI site at the 3′ endof the gene. The PCR product was digested with BamHI to yield a fragmentapproximately 1721 base pairs in length corresponding to the SIV HA(H1N1) gene. Fragment 3 is an approximately 3010 base pair BamHI toPvuII restriction fragment of plasmid pJF751 (11) . Fragment 4 is anapproximately 895 base pair XbaI to HindIII subfragment of the RPVHindIII fragment U (6). The XbaI sites in fragments 1 and 4 wereconverted to unique NotI sites using NotI linkers.

Homology Vector 902-19.18

The plasmid 902-19.18 was constructed for the purpose of insertingforeign DNA into recombinant raccoonpox virus (RPV). It incorporates anE. coli β-galactosidase (lacZ) marker gene and a swine influenza virus(SIV) hemagglutinin (HA) (H1N1) gene flanked by RPV DNA. Upstream of theforeign gene is an approximately 906 base pair fragment of RPV DNA.Downstream of the foreign genes is an approximately 895 base pairfragment of RPV DNA. In contrast to homology vector 902-16.2 directionof transcription of the SIV HA gene is the same as the direction oftranscription of the lacZ gene and the same as the direction oftranscription of the RPV O1L ORF. When the plasmid is used according tothe HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT RPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-galactosidase (lacZ) marker gene is under the control of a latepromoter (LP1) and the SIV HA (H1N1) gene is under the control of thelate/early promoter (LP2EP2). It was constructed utilizing standardrecombinant DNA techniques (20, 21), by joining restriction fragmentsfrom the following sources with the synthetic DNA sequences. The plasmidvector was derived from an approximately 2999 base pair HindIIIrestriction fragment of pSP64 (PROMEGA™). Fragment 1 is an approximately906 base pair HindIII to XbaI restriction sub-fragment of the RPVHindIII restriction fragment U (6). Fragment 2 is an approximately 1721base pair BamHI to BamHI restriction fragment synthesized by reversetranscription and polymerase chain reaction (PCR) using genomic RNA fromthe SIV H1N1 strain (NVSL) To synthesize SIV HA (H1N1), the primer5′-CCGAGGATCCGGCAATACTATTAGTCTTGCTATGTACAT-3′; 6/95.5) (SEQ ID NO 18)synthesized from the 5′ end of the SIV HA (H1N1) gene, introduced anBamHI site at the 5′ end of the gene. The primer(5′-CTCTGGATCCTAATTTAAATACATATTCTGCACTGTA-3′; 6/95.6) (SEQ ID NO 19) wasused for reverse transcription and PCR and synthesized from the 3′ endof the SIV HA (H1N1) gene and introduced a BamHI site at the 3′ end ofthe gene. The PCR product was digested with BamHI to yield a fragmentapproximately 1721 base pairs in length corresponding to the SIV HA(H1N1) gene. Fragment 3 is an approximately 3010 base pair BamHI toPvuII restriction fragment of plasmid pJF751 (11). Fragment 4 is anapproximately 895 base pair XbaI to HindIII subfragment of the RPVHindIII fragment U (6). The XbaI sites in fragments 1 and 4 wereconverted to unique NotI sites using NotI linkers.

Homology Vector 902-49.5

The plasmid 902-49.5 was constructed for the purpose of insertingforeign DNA into recombinant raccoonpox virus (RPV). It incorporates anE. coli β-galactosidase (lacZ) marker gene and a swine influenza virus(SIV) neuraminidase (NA) (H1N1) gene flanked by RPV DNA. Upstream of theforeign gene is an approximately 906 base pair fragment of RPV DNA.Downstream of the foreign genes is an approximately 895 base pairfragment of RPV DNA. When the plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT RPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-galactosidase (lacZ) marker gene is under the control of a latepromoter (LP1) and the SIV NA (H1N1) gene is under the control of thelate/early promoter (LP2EP2). It was constructed utilizing standardrecombinant DNA techniques (20, 21), by joining restriction fragmentsfrom the following sources with the synthetic DNA sequences. The plasmidvector was derived from an approximately 2999 base pair HindIIIrestriction fragment of pSP64 (PROMEGA™). Fragment 1 is an approximately906 base pair HindIII to XbaI restriction sub-fragment of the RPVHindIII restriction fragment U (6). Fragment 2 is an approximately 1414base pair EcoRI to BglII restriction fragment synthesized by reversetranscription and polymerase chain reaction (PCR) using genomic RNA fromthe SIV H1N1 strain (NVSL) To synthesize SIV NA (H1N) the primer5′-AATGAATTCAAAAAATAATAACCATTGGGTCAAT-3′; 6/95.11) (SEQ ID NO 20)synthesized from the 5′ end of the SIV NA (H1N1) gene, introduced anEcoRI site at the 5′ end of the gene. The primer(5′-GGAAGATCTACTTGTCAATGGTGAATGGCAGATCAG-31; 6/95.12) (SEQ ID NO 21) wasused for reverse transcription and PCR and synthesized from the 3′ endof the SIV NA (H1N1) gene and introduced a BglII site at the 3′ end ofthe gene. The PCR product was digested with EcoRI and BglII to yield afragment approximately 1414 base pairs in length corresponding to theSIV NA (H1N1) gene. Fragment 3 is an approximately 3010 base pair BamHIto PvuII restriction fragment of plasmid pJF751 (25). Fragment 4 is anapproximately 895 base pair XbaI to HindIII subfragment of the RPVHindIII fragment U. The XbaI sites in fragments 1 and 4 were convertedto unique NotI sites using NotI linkers.

Homology Vector 902-49.14

The plasmid 902-49.14 was constructed for the purpose of insertingforeign DNA into recombinant raccoonpox virus (RPV). It incorporates anE. coli β-galactosidase (lacZ) marker gene and a porcine reproductiveand respiratory syndrome virus (PRRS) ORF3 gene flanked by RPV DNA.Upstream of the foreign gene is an approximately 906 base pair fragmentof RPV DNA. Downstream of the foreign genes is an approximately 895 basepair fragment of RPV DNA. When the plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT RPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-galactosidase (lacZ) marker gene is under the control of a latepromoter (LP1) and the PRRS ORF3 gene is under the control of thelate/early promoter (LP2EP2). It was constructed utilizing standardrecombinant DNA techniques (20, 21), by joining restriction fragmentsfrom the following sources with the synthetic DNA sequences. The plasmidvector was derived from an approximately 2999 base pair HindIIIrestriction fragment of pSP64 (PROMEGA™). Fragment 1 is an approximately906 base pair HindIII to XbaI restriction sub-fragment of the RPVHindIII restriction fragment U (6). Fragment 2 is an EcoRI to BamHIrestriction fragment synthesized by reverse transcription and polymerasechain reaction (PCR) using genomic RNA from a U.S. Isolate of PRRSobtained from the NVSL (Reference strain, IA-2). To synthesize PRRSORF3, the primer 5′-TTCGAATTCGGCTAATAGCTGTACATTCCTCCATATTT-3′; 1/96.7)(SEQ ID NO 22) synthesized from the 5′ end of the PRRS ORF3 gene,introduced an EcoRI site at the 5′ end of the gene. The primer(5′-GGGGATCCTATCGCCGTACGGCACTGAGGG-3′; 1/96.8) (SEQ ID NO 23) was usedfor reverse transcription and PCR and synthesized from the 3′ end of thePRRS ORF3 gene. The PCR product was digested with EcoRI and BamHI toyield a fragment approximately 768 base pairs in length corresponding tothe PRRS ORF3 gene. Fragment 3 is an approximately 3010 base pair BamHIto PvuII restriction fragment of plasmid pJF751 (25). Fragment 4 is anapproximately 895 base pair XbaI to HindIII subfragment of the RPVHindIII fragment U. The XbaI sites in fragments 1 and 4 were convertedto unique NotI sites using NotI linkers.

Homology Vector 902-49.23

The plasmid 902-49.23 was constructed for the purpose of insertingforeign DNA into recombinant raccoonpox virus (RPV). It incorporates anE. coli β-galactosidase (lacZ) marker gene and a porcine reproductiveand respiratory syndrome virus (PRRS) ORF4 gene flanked by RPV DNA.Upstream of the foreign gene is an approximately 906 base pair fragmentof RPV DNA. Downstream of the foreign genes is an approximately 895 basepair fragment of RPV DNA. When the plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT RPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-galactosidase (lacZ) marker gene is under the control of a latepromoter (LP1) and the PRRS ORF4 gene is under the control of thelate/early promoter (LP2EP2). It was constructed utilizing standardrecombinant DNA techniques (20, 21), by joining restriction fragmentsfrom the following sources with the synthetic DNA sequences. The plasmidvector was derived from an approximately 2999 base pair HindIIIrestriction fragment of pSP64 (PROMEGA™). Fragment 1 is an approximately906 base pair HindIII to XbaI restriction sub-fragment of the RPVHindIII restriction fragment U (6). Fragment 2 is an EcoRI to BamHIrestriction fragment synthesized by reverse transcription and polymerasechain reaction (PCR) using genomic RNA from a U.S. Isolate of PRRSobtained from the NVSL (Reference strain, IA-2). To synthesize PRRSORF4, the primer (5′-CCGAATTCGGCTGCGTCCCTTCTTTTCCTCATGG-3′; 1/96.11)(SEQ ID NO 24) synthesized from the 5′ end of the PRRS ORF4 gene,introduced an EcoRI site at the 5′ end of the gene. The primer(5μ′CTGGATCCTTCAAATTGCCAACAGAATGGCAAAAAGAC-3′; 1/96.12) (SEQ ID NO 25)was used for reverse transcription and PCR and synthesized from the 3′end of the PRRS ORF4 gene. The PCR product was digested with EcoRI andBamHI to yield a fragment approximately 542 base pairs in lengthcorresponding to the PRRS ORF4 gene. Fragment 3 is an approximately 3010base pair BamHI to PvuII restriction fragment of plasmid pJF751 (25).Fragment 4 is an approximately 895 base pair XbaI to HindIII subfragmentof the RPV HindIII fragment U. The XbaI sites in fragments 1 and 4 wereconverted to unique NotI sites using NotI linkers.

Homology Vector 902-49.34

The plasmid 902-49.34 was constructed for the purpose of insertingforeign DNA into RPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and a porcine reproductive and respiratory syndrome virus(PRRS) ORF5 gene flanked by RPV DNA. Upstream of the foreign gene is anapproximately 906 base pair fragment of RPV DNA. Downstream of theforeign genes is an approximately 895 base pair fragment of RPV DNA.When the plasmid is used according to the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT RPV, a virus containing DNA codingfor the foreign genes will result. Note that the β-galactosidase (lacZ)marker gene is under the control of a late promoter (LP1) and the PRRSORF5 gene is under the control of the late/early promoter (LP2EP2). Itwas constructed utilizing standard recombinant DNA techniques (20, 21),by joining restriction fragments from the following sources with thesynthetic DNA sequences. The plasmid vector was derived from anapproximately 2999 base pair HindIII restriction fragment of pSP64(PROMEGA™). Fragment 1 is an approximately 906 base pair HindIII to XbaIrestriction sub-fragment of the RPV HindIII restriction fragment U (6).Fragment 2 is an EcoRI to BamHI restriction fragment synthesized byreverse transcription and polymerase chain reaction (PCR) using genomicRNA from a U.S. Isolate of PRRS obtained from the NVSL (Referencestrain, IA-2). To synthesize PRRS ORF5, the primer(5′-TTGAATTCGTTGGAGAAATGCTTGACCGCGGGC-3′; 1/96.13) (SEQ ID NO 26)synthesized from the 5′ end of the PRRS ORF5 gene, introduced an EcoRIsite at the 5′ end of the gene. The primer(5′-GAAGGATCCTAAGGACGACCCCATTGTTCCGCTG-3′; 1/96.14) (SEQ ID NO 27) wasused for reverse transcription and PCR and synthesized from the 3′ endof the PRRS ORF5 gene. The PCR product was digested with EcoRI and BamHIto yield a fragment approximately 606 base pairs in length correspondingto the PRRS ORF5 gene. Fragment 3 is an approximately 3010 base pairBamHI to PvuII restriction fragment of plasmid pJF751 (25). Fragment 4is an approximately 895 base pair XbaI to HindIII subfragment of the RPVHindIII fragment U. The XbaI sites in fragments 1 and 4 were convertedto unique NotI sites using NotI linkers.

Homology Vector 902-49.46

The plasmid 902-49.46 was constructed for the purpose of insertingforeign DNA into RPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene flanked by RPV DNA. Upstream of the foreign gene is anapproximately 906 base pair fragment of RPV DNA. Downstream of theforeign genes is an approximately 895 base pair fragment of RPV DNA.When the plasmid is used according to the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT RPV, a virus containing DNA codingfor the foreign genes will result. Note that the β-galactosidase (lacZ)marker gene is under the control of a late promoter (LP1) and a secondforeign DNA is inserted into an EcoRI or BamHI site, and the secondforeign DNA is under the control of the late/early promoter (LP2EP2). Itwas constructed utilizing standard recombinant DNA techniques (20, 21),by joining restriction fragments from the following sources with thesynthetic DNA sequences. The plasmid vector was derived from anapproximately 2999 base pair HindIII restriction fragment of pSP64(PROMEGA™). Fragment 1 is an approximately 906 base pair HindIII to XbaIrestriction sub-fragment of the RPV HindIII restriction fragment U (6).Fragment 2 is an approximately 3010 base pair BamHI to PvuII restrictionfragment of plasmid pJF751 (25). Fragment 3 is an approximately 895 basepair XbaI to HindIII subfragment of the RPV HindIII fragment U. The XbaIsites in fragments 1 and 3 were converted to unique NotI sites usingNotI linkers. Synthetic DNA between fragments 2 and 3 contains theLP2EP2 promoter and an EcoRI site and a BamHI site for insertion offoreign DNA. (See FIG. 6).

Homology Vector 902-67.1

The plasmid 902-67.1 was constructed for the purpose of insertingforeign DNA into RPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene flanked by RPV DNA. Upstream of the foreign gene is anapproximately 1500 base pair fragment of RPV DNA. Downstream of theforeign genes is an approximately 3600 base pair fragment of RPV DNA.When the plasmid is used according to the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT RPV, a virus containing DNA codingfor the foreign genes will result. Note that the β-galactosidase (lacZ)marker gene is under the control of a late promoter (LP1) and a secondforeign DNA is inserted into an EcoRI or BamHI site, and the secondforeign DNA is under the control of the late/early promoter (LP2EP2). Itwas constructed utilizing standard recombinant DNA techniques (20, 21),by joining restriction fragments from the following sources with thesynthetic DNA sequences. The plasmid vector was derived from anapproximately 2999 base pair HindIII restriction fragment of pSP64(PROMEGA™). Fragment 1 is an approximately 2200 base pair HindIII toSnaBI restriction sub-fragment of the RPV HindIII restriction fragment N(6). Fragment 2 is an approximately 3010 base pair BamHI to PvuIIrestriction fragment of plasmid pJF751 (25). Fragment 3 is anapproximately 2900 base pair SnaBI to HindIII subfragment of the RPVHindIII fragment N. The SnaB I sites in fragments 1 and 3 were convertedto unique NotI sites using NotI linkers. Synthetic DNA between fragments2 and 3 contains the LP2EP2 promoter and an EcoRI site and a BamHI sitefor insertion of foreign DNA. (See FIG. 6).

Homology Vector 902-67.14

The plasmid 902-67.14 was constructed for the purpose of insertingforeign DNA into RPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene flanked by RPV DNA. Upstream of the foreign gene is anapproximately 1500 base pair fragment of RPV DNA. Downstream of theforeign genes is an approximately 3600 base pair fragment of RPV DNA.When the plasmid is used according to the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT RPV, a virus containing DNA codingfor the foreign genes will result. Note that the β-galactosidase (lacZ)marker gene is under the control of a late promoter (LP1) and a secondforeign DNA is inserted into an EcoRI or BamHI site, and the secondforeign DNA is under the control of the late/early promoter (LP2EP2). Itwas constructed utilizing standard recombinant DNA techniques (20, 21),by joining restriction fragments from the following sources with thesynthetic DNA sequences. The plasmid vector was derived from anapproximately 2999 base pair HindIII restriction fragment of pSP64(PROMEGA™). Fragment 1 is an approximately 1500 base pair HindIII toEcoRV restriction sub-fragment of the RPV HindIII restriction fragment N(6). Fragment 2 is an approximately 3010 base pair BamHI to PvuIIrestriction fragment of plasmid pJF751 (25). Fragment 3 is anapproximately 3600 base pair EcoRV to HindIII subfragment of the RPVHindIII fragment N. The EcoRV sites in fragments 1 and 3 were convertedto unique NotI sites using NotI linkers. Synthetic DNA between fragments2 and 3 contains the LP2EP2 promoter and an EcoRI site and a BamHI sitefor insertion of foreign DNA. (See FIG. 6).

Homology Vector 902-67.27

The plasmid 902-67.27 was constructed for the purpose of insertingforeign DNA into RPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene flanked by RPV DNA. Upstream of the foreign gene is anapproximately 2200 base pair fragment of RPV DNA. Downstream of theforeign genes is an approximately 2900 base pair fragment of RPV DNA.When the plasmid is used according to the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT RPV, a virus containing DNA codingfor the foreign genes will result. Note that the β-galactosidase (lacZ)marker gene is under the control of a late promoter (LP1) and a secondforeign DNA is inserted into an EcoRI or BamHI site, and the secondforeign DNA is under the control of the late/early promoter (LP2EP2). Itwas constructed utilizing standard recombinant DNA techniques (20, 21),by joining restriction fragments from the following sources with thesynthetic DNA sequences. The plasmid vector was derived from anapproximately 2999 base pair HindIII restriction fragment of pSP64(PROMEGA™). Fragment 1 is an approximately 2200 base pair HindIII toEcoRV restriction sub-fragment of the RPV HindIII restriction fragment N(6). Fragment 2 is an approximately 3010 base pair BamHI to PvuIIrestriction fragment of plasmid pJF751 (25). Fragment 3 is anapproximately 2900 base pair EcoRV to HindIII subfragment of the RPVHindIII fragment N. The EcoRV sites in fragments 1 and 3 were convertedto unique NotI sites using NotI linkers. Synthetic DNA between fragments2 and 3 contains the LP2EP2 promoter and an EcoRI site and a BamHI sitefor insertion of foreign DNA. (See FIG. 6).

Homology Vector 919-16.12

The plasmid 919-16.12 was constructed for the purpose of insertingforeign DNA into RPV. It incorporates an E. coli β-glucuronidase (uidA)marker gene flanked by RPV DNA. Upstream of the foreign gene is anapproximately 906 base pair fragment of RPV DNA. Downstream of theforeign genes is an approximately 895 base pair fragment of RPV DNA.When the plasmid is used according to the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT RPV, a virus containing DNA codingfor the foreign genes will result. Note that the β-glucuronidase (uidA)marker gene is under the control of a late promoter (LP1) and a secondforeign DNA is inserted into an EcoRI or BamHI site, and the foreign DNAis under the control of the late/early promoter (LP2EP2). It wasconstructed utilizing standard recombinant DNA techniques (20, 21), byjoining restriction fragments from the following sources with thesynthetic DNA sequences. The plasmid vector was derived from anapproximately 2999 base pair HindIII restriction fragment of pSP64(PROMEGA™). Fragment 1 is an approximately 906 base pair HindIII to XbaIrestriction sub-fragment of the RPV HindIII restriction fragment U (6).Fragment 2 is an approximately 1823 base pair NotI restriction fragmentof plasmid pRAJ260 (CLONETECH™). Fragment 3 is an approximately 895 basepair XbaI to HindIII subfragment of the RPV HindIII fragment U. The XbaIsites in fragments 1 and 3 were converted to unique NotI sites usingNotI linkers. Synthetic DNA between fragments 2 and 3 contains theLP2EP2 promoter and an EcoRI site and a BamHI site for insertion offoreign DNA. (See FIG. 5).

Homology Vector 919-63.21

The plasmid 919-63.21 was constructed for the purpose of insertingforeign DNA into RPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and a porcine parvovirus (PPV) VP2 capsid protein geneflanked by RPV DNA. Upstream of the foreign gene is an approximately 906base pair fragment of RPV DNA. Downstream of the foreign genes is anapproximately 895 base pair fragment of RPV DNA. When the plasmid isused according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT RPV, a virus containing DNA coding for the foreign geneswill result. Note that the β-galactosidase (lacZ) marker gene is underthe control of a late promoter (LP1) and the PPV VP2 gene is under thecontrol of the late/early promoter (LP2EP2). It was constructedutilizing standard recombinant DNA techniques (20, 21), by joiningrestriction fragments from the following sources with the synthetic DNAsequences. The plasmid vector was derived from an approximately 2999base pair HindIII restriction fragment of pSP64 (PROMEGA™). Fragment 1is an approximately 906 base pair HindIII to XbaI restrictionsub-fragment of the RPV HindIII restriction fragment U (6). Fragment 2is a BamHI restriction fragment synthesized by reverse transcription andpolymerase chain reaction (PCR) using genomic RNA from a U.S. Isolate ofPPV obtained from the NADL (Strain NADL-2; non-pathogenic vaccinestrain). To synthesize PPV VP2, the primer(5′-CTAAGGATCCGAGTGAAAATGTGGAACAACACAACCC-3′; 4/97.50) (SEQ ID NO 31)synthesized from the 5′ end of the PPV VP2 gene, introduced an BamHIsite at the 5′ end of the gene. The primer(5′-GTGGATCCTAGTATAATTTTCTTGGTATAAGTTGTGAA-3′; 4/97.51) (SEQ ID NO 32)was used for reverse transcription and PCR and synthesized from the 3′end of the PPV VP2 gene. The PCR product was digested with BamHI toyield a fragment approximately 1700 base pairs in length correspondingto the PPV VP2 gene. Fragment 3 is an approximately 3010 base pair BamHIto PvuII restriction fragment of plasmid pJF751 (25). Fragment 4 is anapproximately 895 base pair XbaI to HindIII subfragment of the RPVHindIII fragment U. The XbaI sites in fragments 1 and 4 were convertedto unique NotI sites using NotI linkers.

Homology Vector 934-64.2

The plasmid 934-64.2 was constructed for the purpose of insertingforeign DNA into RPV. It incorporates an E. coli β-glucuronidase (uidA)marker gene and the gene for porcine reproductive and respiratorysyndrome virus (PRRS) ORF3 (Eichelberger strain) flanked by RPV DNA.Upstream of the foreign genes is an approximately 906 base pair fragmentof RPV DNA. Downstream of the foreign genes is an approximately 895 basepair fragment of RPV DNA. When the plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT RPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-glucuronidase (uidA) marker gene is under the control of a latepromoter (LP1) and the PRRS ORF3 is inserted into an EcoRI and BamHIsite, and is under the control of the early promoter (EP2). It wasconstructed utilizing standard recombinant DNA techniques (20, 21), byjoining restriction fragments from the following sources with thesynthetic DNA sequences. The plasmid vector was derived from anapproximately 2999 base pair HindIII restriction fragment of pSP64(PROMEGA™). Fragment 1 is an approximately 906 base pair HindIII to XbaIrestriction sub-fragment of the RPV HindIII restriction fragment U (6).Fragment 2 is an approximately 1823 base pair NotI restriction fragmentof plasmid pRAJ260 (CLONETECH™). Fragment 3 is an EcoRI restrictionfragment synthesized by reverse transcription and polymerase chainreaction (PCR) using genomic RNA from a U.S. isolate of PRRS obtainedfrom the Eichelberger atypical strain. To synthesize PRRS ORF3, theprimer 5′-CAATGAATTCTAGCTGTGCACTCCTCCATATTTTCCTC-3′; 9/97.9) (SEQ ID NO45) synthesized from the 5′ end of the PRRS ORF3 gene, introduced anEcoRI site at the 5′ end of the gene. The primer(5′-CGGGAATTCCTATCGCCGTACGGCACTGAGG-3′; 9/97.10) (SEQ ID NO 46) was usedfor reverse transcription and PCR and synthesized from the 3′ end of thePRRS ORF3 gene, introduced an EcoRI site at the 3′ end of the gene. ThePCR product was digested with EcoRI and BamHI to yield a fragmentapproximately 768 base pairs in length corresponding to the PRRS ORF3gene.

Fragment 4 is an approximately 895 base pair XbaI to HindIII subfragmentof the RPV HindIII fragment U. The XbaI sites in fragments 1 and 3 wereconverted to unique NotI sites using NotI linkers.

Homology Vector 934-65.1

The plasmid 934-65.1 was constructed for the purpose of insertingforeign DNA into RPV. It incorporates an E. coli β-glucuronidase (uidA)marker gene and the gene for porcine reproductive and respiratorysyndrome virus (PRRS) ORF2 (Eichelberger strain) flanked by RPV DNA.Upstream of the foreign genes is an approximately 906 base pair fragmentof RPV DNA. Downstream of the foreign genes is an approximately 895 basepair fragment of RPV DNA. When the plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT RPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-glucuronidase (uidA) marker gene is under the control of a latepromoter (LP1) and the PRRS ORF2 is inserted into an EcoRI and BamHIsite, and is under the control of the early promoter (EP2). It wasconstructed utilizing standard recombinant DNA techniques (20, 21), byjoining restriction fragments from the following sources with thesynthetic DNA sequences. The plasmid vector was derived from anapproximately 2999 base pair HindIII restriction fragment of pSP64(PROMEGA™). Fragment 1 is an approximately 906 base pair HindIII to XbaIrestriction sub-fragment of the RPV HindIII restriction fragment U (6).Fragment 2 is an approximately 1823 base pair NotI restriction fragmentof plasmid pRAJ260 (CLONETECH™). Fragment 3 is an EcoRI restrictionfragment synthesized by reverse transcription and polymerase chainreaction (PCR) using genomic RNA from a U.S. Isolate of PRRS obtainedfrom the Eichelberger atypical strain. To synthesize PRRS ORF2, theprimer 5′-CTGAGAATTCATGGGGGCTATGCAAAGCCTTTTCG-3′; 9/97.13) (SEQ ID NO43) synthesized from the 5′ end of the PRRS ORF2 gene, introduced anEcoRI site at the 5′ end of the gene. The primer(5′-GTGGAATTCACCGTGAGTTCGAAAGAAAAATTGC-3′; 9/97.14) (SEQ ID NO 44) wasused for reverse transcription and PCR and synthesized from the 3′ endof the PRRS ORF2 gene, introduced an EcoRI site at the 3′ end of thegene. The PCR product was digested with EcoRI to yield a fragmentapproximately 771 base pairs in length corresponding to the PRRS ORF2gene. Fragment 4 is an approximately 895 base pair XbaI to HindIIIsubfragment of the RPV HindIII fragment U. The XbaI sites in fragments 1and 3 were converted to unique NotI sites using NotI linkers.

Homology Vector 935-50.1

The plasmid 935-50.1 was constructed for the purpose of insertingforeign DNA into RPV. It incorporates an E. coli β-glucuronidase (uidA)marker gene flanked by RPV DNA. Upstream of the foreign gene is anapproximately 906 base pair fragment of RPV DNA. Downstream of theforeign genes is an approximately 895 base pair fragment of RPV DNA.When the plasmid is used according to the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT RPV, a virus containing DNA codingfor the foreign genes will result. Note that the β-glucuronidase (uidA)marker gene is under the control of a late promoter (LP1). It wasconstructed utilizing standard recombinant DNA techniques (20, 21), byjoining restriction fragments from the following sources with thesynthetic DNA sequences. The plasmid vector was derived from anapproximately 2999 base pair HindIII restriction fragment of pSP64(PROMEGA™). Fragment 1 is an approximately 906 base pair HindIII to XbaIrestriction sub-fragment of the RPV HindIII restriction fragment U (6).Fragment 2 is an approximately 1823 base pair NotI restriction fragmentof plasmid pRAJ260 (CLONETECH™). Fragment 3 is an approximately 895 basepair XbaI to HindIII subfragment of the RPV HindIII fragment U. The XbaIsites in fragments 1 and 3 were converted to unique NotI sites usingNotI linkers.

Homology Vector 936-87.2

The plasmid 936-87.2 was constructed for the purpose of insertingforeign DNA into RPV. It incorporates an E. coli β-glucuronidase (uidA)marker gene and the gene for porcine reproductive and respiratorysyndrome virus (PRRS) ORF7 (Eichelberger strain) flanked by RPV DNA.Upstream of the foreign genes is an approximately 906 base pair fragmentof RPV DNA. Downstream of the foreign genes is an approximately 895 basepair fragment of RPV DNA. When the plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT RPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-glucuronidase (uidA) marker gene is under the control of a latepromoter (LP1) and the PRRS ORF7 is inserted into an EcoRI and BamHIsite, and is under the control of the early promoter (EP2). It wasconstructed utilizing standard recombinant DNA techniques (20, 21), byjoining restriction fragments from the following sources with thesynthetic DNA sequences. The plasmid vector was derived from anapproximately 2999 base pair HindIII restriction fragment of pSP64(PROMEGA™). Fragment 1 is an approximately 906 base pair HindIII to XbaIrestriction sub-fragment of the RPV HindIII restriction fragment U (6).Fragment 2 is an approximately 1823 base pair NotI restriction fragmentof plasmid pRAJ260 (CLONETECH™). Fragment 3 is an EcoRI to BamHIrestriction fragment synthesized by reverse transcription and polymerasechain reaction (PCR) using genomic RNA from a U.S. Isolate of PRRSobtained from the Eichelberger atypical strain. To synthesize PRRS ORF7,the primer 5′-TATGAATTCTAACAACGGCAAGCAGCAAAAGAAAAAG-3′; 9/97.15) (SEQ IDNO 53) synthesized from the 5′ end of the PRRS ORF7 gene, introduced anEcoRI site at the 5′ end of the gene. The primer (5μ′-TGTGGATCCATCACGCTGTGGGTGATGCTGTAG-3′; 9/97.16) (SEQ ID NO 54) was usedfor reverse transcription and PCR and synthesized from the 3′ end of thePRRS ORF7 gene, introduced an BamHI site at the 3′ end of the gene. ThePCR product was digested with EcoRI and BamHI to yield a fragmentapproximately 372 base pairs in length corresponding to the PRRS ORF7gene.

Fragment 4 is an approximately 895 base pair XbaI to HindIII subfragmentof the RPV HindIII fragment U. The XbaI sites in fragments 1 and 3 wereconverted to unique NotI sites using NotI linkers.

Homology Vector 938-94.1

The plasmid 938-94.1 was constructed for the purpose of insertingforeign DNA into RPV. It incorporates an E. coli β-glucuronidase (uidA)marker gene and the gene for porcine reproductive and respiratorysyndrome virus (PRRS) ORF4 (Eichelberger strain) flanked by RPV DNA.Upstream of the foreign genes is an approximately 906 base pair fragmentof RPV DNA. Downstream of the foreign genes is an approximately 895 basepair fragment of RPV DNA. When the plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT RPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-glucuronidase (uidA) marker gene is under the control of a latepromoter (LP1) and the PRRS ORF4 is inserted into an EcoRI and BamHIsite, and is under the control of the early promoter (EP2). It wasconstructed utilizing standard recombinant DNA techniques (20, 21), byjoining restriction fragments from the following sources with thesynthetic DNA sequences. The plasmid vector was derived from anapproximately 2999 base pair HindIII restriction fragment of pSP64(PROMEGA™). Fragment 1 is an approximately 906 base pair HindIII to XbaIrestriction sub-fragment of the RPV HindIII restriction fragment U (6).Fragment 2 is an approximately 1823 base pair NotI restriction fragmentof plasmid pRAJ260 (CLONETECH™). Fragment 3 is an EcoRI to BamHIrestriction fragment synthesized by reverse transcription and polymerasechain reaction (PCR) using genomic RNA from a U.S. Isolate of PRRSobtained from the Eichelberger atypical strain. To synthesize PRRS ORF4,the primer 5′-GGGAATTCTGCGTCCCTTCTTTTCCTCTTGGTTG-3′; 9/97.7) (SEQ ID NO47) synthesized from the 5′ end of the PRRS ORF4 gene, introduced anEcoRI site at the 5′ end of the gene. The primer(5′-CTTGGATCCTCAAATTGCCAGCAGGATGGCAAAAAG-3′; 9/97.8) (SEQ ID NO 48) wasused for reverse transcription and PCR and synthesized from the 3′ endof the PRRS ORF4 gene, introduced an BamHI site at the 3′ end of thegene. The PCR product was digested with EcoRI to yield a fragmentapproximately 542 base pairs in length corresponding to the PRRS ORF4gene.

Fragment 4 is an approximately 895 base pair XbaI to HindIII subfragmentof the RPV HindIII fragment U. The XbaI sites in fragments 1 and 3 wereconverted to unique NotI sites using NotI linkers.

Homology Vector 938-94.25

The plasmid 938-94.25 was constructed for the purpose of insertingforeign DNA into RPV. It incorporates an E. coli β-glucuronidase (uidA)marker gene and the gene for porcine reproductive and respiratorysyndrome virus (PRRS) ORF5 (Eichelberger strain) flanked by RPV DNA.Upstream of the foreign genes is an approximately 906 base pair fragmentof RPV DNA. Downstream of the foreign genes is an approximately 895 basepair fragment of RPV DNA. When the plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT RPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-glucuronidase (uidA) marker gene is under the control of a latepromoter (LP1) and the PRRS ORF5 is inserted into an EcoRI and BamHIsite, and is under the control of the early promoter (EP2). It wasconstructed utilizing standard recombinant DNA techniques (20, 21), byjoining restriction fragments from the following sources with thesynthetic DNA sequences. The plasmid vector was derived from anapproximately 2999 base pair HindIII restriction fragment of pSP64(PROMEGA™). Fragment 1 is an approximately 906 base pair HindIII to XbaIrestriction sub-fragment of the RPV HindIII restriction fragment U (6).Fragment 2 is an approximately 1823 base pair NotI restriction fragmentof plasmid pRAJ260 (CLONETECH™). Fragment 3 is an EcoRI to BamHIrestriction fragment synthesized by reverse transcription and polymerasechain reaction (PCR) using genomic RNA from a U.S. Isolate of PRRSobtained from the Eichelberger atypical strain. To synthesize PRRS ORF5,the primer 5′-CAAGGAATTCGGGGAAATGCTTGACCGCGGGCTG-3′; 9/97.5) (SEQ ID NO49) synthesized from the 5′ end of the PRRS ORF5 gene, introduced anEcoRI site at the 5′ end of the gene. The primer(5′-AAAAGGATCCTAGGGACGACCCCATTGTTCAGC-3′; 9/97.6) (SEQ ID NO 50) wasused for reverse transcription and PCR and synthesized from the 3′ endof the PRRS ORF5 gene, introduced an BamHI site at the 3′ end of thegene. The PCR product was digested with EcoRI and BamHI to yield afragment approximately 606 base pairs in length corresponding to thePRRS ORF5 gene.

Fragment 4 is an approximately 895 base pair XbaI to HindIII subfragmentof the RPV HindIII fragment U. The XbaI sites in fragments 1 and 3 wereconverted to unique NotI sites using NotI linkers.

Homology Vector 950-13.4

The plasmid 950-13.4 was constructed for the purpose of insertingforeign DNA into RPV. It incorporates an E. coli β-glucuronidase (uidA)marker gene and the gene for porcine reproductive and respiratorysyndrome virus (PRRS) ORF6 (Eichelberger strain) flanked by RPV DNA.Upstream of the foreign genes is an approximately 906 base pair fragmentof RPV DNA. Downstream of the foreign genes is an approximately 895 basepair fragment of RPV DNA. When the plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT RPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-glucuronidase (uidA) marker gene is under the control of a latepromoter (LP1) and the PRRS ORF6 is inserted into an EcoRI and BamHIsite, and is under the control of the early promoter (EP2). It wasconstructed utilizing standard recombinant DNA techniques (20, 21), byjoining restriction fragments from the following sources with thesynthetic DNA sequences. The plasmid vector was derived from anapproximately 2999 base pair HindIII restriction fragment of pSP64(PROMEGA™). Fragment 1 is an approximately 906 base pair HindIII to XbaIrestriction sub-fragment of the RPV HindIII restriction fragment U (6).Fragment 2 is an approximately 1823 base pair NotI restriction fragmentof plasmid pRAJ260 (CLONETECH™). Fragment 3 is an EcoRI to BamHIrestriction fragment synthesized by reverse transcription and polymerasechain reaction (PCR) using genomic RNA from a U.S. Isolate of PRRSobtained from the Eichelberger atypical strain. To synthesize PRRS ORF6,the primer 5′-CAATGAATTCGTCCCTAGACGACTTTTGCAATGATAG-3′; 9/97.5) (SEQ IDNO 51) synthesized from the 5′ end of the PRRS ORF6 gene, introduced anEcoRI site at the 5′ end of the gene. The primer(5′-CTTGGGATCCTTATTTGGCATATTTGACAAGGTTTACCAC-3′; 9/97.6) (SEQ ID NO 52)was used for reverse transcription and PCR and synthesized from the 3′end of the PRRS ORF6 gene, introduced an BamHI site at the 3′ end of thegene. The PCR product was digested with EcoRI and BamHI to yield afragment approximately 525 base pairs in length corresponding to thePRRS ORF6 gene.

Fragment 4 is an approximately 895 base pair XbaI to HindIII subfragmentof the RPV HindIII fragment U. The XbaI sites in fragments 1 and 3 wereconverted to unique NotI sites using NotI linkers.

Homology Vector 950-16.34

The plasmid 950-16.34 was constructed for the purpose of insertingforeign DNA into RPV. It incorporates an E. coli β-glucuronidase (uidA)marker gene and the gene for porcine reproductive and respiratorysyndrome virus (PRRS) ORF5 and ORF6 (Eichelberger strain) flanked by RPVDNA. Upstream of the foreign genes is an approximately 906 base pairfragment of RPV DNA. Downstream of the foreign genes is an approximately895 base pair fragment of RPV DNA. When the plasmid is used according tothe HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT RPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-glucuronidase (uidA) marker gene is under the control of a latepromoter (LP1), the PRRS ORF5 is inserted into an EcoRI and BamHI site,and is under the control of the early promoter (EP2), and PRRS ORF6 isinserted into an EcoRI and BamHI site, and is under the control of thelate promoter (LP2),. It was constructed utilizing standard recombinantDNA techniques (20, 21), by joining restriction fragments from thefollowing sources with the synthetic DNA sequences. The plasmid vectorwas derived from an approximately 2999 base pair HindIII restrictionfragment of pSP64 (PROMEGA™). Fragment 1 is an approximately 906 basepair HindIII to XbaI restriction sub-fragment of the RPV HindIIIrestriction fragment U (6). Fragment 2 is an approximately 1823 basepair NotI restriction fragment of plasmid pRAJ260 (CLONETECH™). Fragment3 is an EcoRI to BamHI restriction fragment synthesized by reversetranscription and polymerase chain reaction (PCR) using genomic RNA froma U.S. Isolate of PRRS obtained from the Eichelberger atypical strain.To synthesize PRRS ORF5, the primer5′-CAAGGAATTCGGGGAAATGCTTGACCGCGGGCTG-3′; 9/97.5) (SEQ ID NO 49)synthesized from the 5′ end of the PRRS ORF5 gene, introduced an EcoRIsite at the 5′ end of the gene. The primer(5′-AAAAGGATCCTAGGGACGACCCCATTGTTCAGC-3′; 9/97.6) (SEQ ID NO 50) wasused for reverse transcription and PCR and synthesized from the 3′ endof the PRRS ORF5 gene, introduced an BamHI site at the 3′ end of thegene. The PCR product was digested with EcoRI and BamHI to yield afragment approximately 606 base pairs in length corresponding to thePRRS ORF5 gene.

Fragment 4 is an EcoRI to BamHI restriction fragment synthesized byreverse transcription and polymerase chain reaction (PCR) using genomicRNA from a U.S. Isolate of PRRS obtained from the Eichelberger atypicalstrain. To synthesize PRRS ORF6, the primer5′-CAATGAATTCGTCCCTAGACGACTTTTGCAATGATAG-3′; 9/97.5) (SEQ ID NO 51)synthesized from the 5′ end of the PRRS ORF6 gene, introduced an EcoRIsite at the 5′ end of the gene. The primer(5′-CTTGGGATCCTTATTTGGCATATTTGACAAGGTTTACCAC-3′; 9/97.6) (SEQ ID NO 52)was used for reverse transcription and PCR and synthesized from the 3′end of the PRRS ORF6 gene, introduced an BamHI site at the 3′ end of thegene. The PCR product was digested with EcoRI and BamHI to yield afragment approximately 525 base pairs in length corresponding to thePRRS ORF6 gene.

Fragment 5 is an approximately 895 base pair XbaI to HindIII subfragmentof the RPV HindIII fragment U. The XbaI sites in fragments 1 and 3 wereconverted to unique NotI sites using NotI linkers.

Homology Vector for S-RPV-014

The plasmid is constructed for the purpose of inserting foreign DNA intoRPV. It incorporates an E. coli β-galactosidase (lacZ) marker geneflanked by RPV DNA. Upstream of the foreign gene is an approximately 906base pair fragment of RPV DNA. Downstream of the foreign genes is anapproximately 895 base pair fragment of RPV DNA. When the plasmid isused according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT RPV, a virus containing DNA coding for the foreign geneswill result. Note that the β-galactosidase (lacZ) marker gene is underthe control of a late promoter, swinepox virus I5L (FIG. 9). It isconstructed utilizing standard recombinant DNA techniques (20, 21), byjoining restriction fragments from the following sources with thesynthetic DNA sequences. The plasmid vector is derived from anapproximately 2999 base pair HindIII restriction fragment of pSP64(PROMEGA™). Fragment 1 is an approximately 906 base pair HindIII to XbaIrestriction sub-fragment of the RPV HindIII restriction fragment U (6).Fragment 2 is an 3010 base pair BamHI to PvuII restriction fragment ofplasmid pJF751 (25). Fragment 3 is an approximately 895 base pair XbaIto HindIII subfragment of the RPV HindIII fragment U. The XbaI sites infragments 1 and 3 were converted to unique NotI sites using NotIlinkers.

EXAMPLE 1

Recombinant raccoonpox virus (RPV) capable of replication which containsforeign DNA encoding an antigenic polypeptide is useful to preventdisease in porcine, bovine, sheep, goats, ovine, caprine, feline,canine, avian, equine, and human species. Useful insertion sites forforeign DNA in non-essential regions in the raccoonpox virus genome arein genomic fragments HindIII U (2.1 kb), HindIII M (6.0 kb), HindIII N(5.1 kb), HindIII W (1.5 kb), HindIII T (2.2 kb, HindIII P (4.5 kb).Open reading frames in each of these fragments which are suitable forinsertion of foreign DNA include, but are not limited to: O1L homolog,E11L homolog in HindIII U; D1L homolog, C17L/B23R homolog in HindIII M;D1L homolog, B22R homolog in HindIII N; N1L homolog, N2L homolog, M1Lhomolog, M2L homolog, K1L homolog, K2L homolog, K7R homolog in HindIIIW, T, or P.

EXAMPLE 2

S-RPV-001

S-RPV-001 is a raccoonpox virus that expresses at least two foreigngenes. The gene for E. coli β-galactosidase (lacZ) and the gene forswine influenza virus (SIV) HA (H1N1) were inserted into the RPV O1L ORF(a unique NotI restriction site has replaced an XbaI restriction site)within the RPV HindIII “U” genomic fragment. The lacZ gene is under thecontrol of the synthetic late promoter (LP1), and the SIV HA gene isunder the control of the synthetic late/early promoter (LP2EP2).Transcription of the SIV HA gene is opposite to the direction oftranscription of the lacZ gene and opposite to the direction oftranscription of the RPV O1L ORF.

S-RPV-001 was derived from S-RPV-000 (ATCC Strain VR-838). This wasaccomplished utilizing the homology vector 902-16.2 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-galactosidase (BLUOGAL™ ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-RPV-001. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-RPV-001 was assayed for expression of SIV-specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT RPV.Polyclonal swine anti-SIV serum or polyclonal goat anti-HA serum wasshown to react specifically with S-RPV-001 plaques and not withS-RPV-000 negative control plaques. All S-RPV-001 observed plaquesreacted with the antiserum indicating that the virus was stablyexpressing the SIV HA protein.

To confirm the expression of the SIV HA protein gene product, cells wereinfected with S-RPV-001 and samples of infected cell lysates and culturesupernatants were subjected to SDS polyacrylamide gel electrophoresis.The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE.Polyclonal swine anti-SIV serum or polyclonal goat anti-HA serum wasused to detect expression of SIV specific proteins. The cell lysate andculture supernatant from S-RPV-001 infected cells exhibited bandscorresponding to 64 kd, which is the expected size of the SIV HAprotein.

S-RPV-001 is useful as a vaccine in swine against swine influenzainfection. S-RPV-001 is useful as a vaccine in combination withS-RPV-003 which expresses SIV NA. S-RPV-001 is also useful forexpression of the SIV HA protein.

EXAMPLE 3

S-RPV-002

S-RPV-002 (ATCC Accession No. VR-2597) is a raccoonpox virus (RPV) thatexpresses at least two foreign genes. The gene for E. coliβ-galactosidase (lacZ) and the gene for swine influenza virus (SIV) HA(H1N1) were inserted into the RPV O1L ORF (a unique NotI restrictionsite has replaced an XbaI restriction site) within the RPV HindIII “U”genomic fragment. The lacZ gene is under the control of the syntheticlate promoter (LP1), and the SIV HA gene is under the control of thesynthetic late/early promoter (LP2EP2). In contrast to S-RPV-001,direction of transcription of the SIV HA gene in S-RPV-002 is the sameas the direction of transcription of the lacZ gene and the direction oftranscription of the RPV O1L ORF.

S-RPV-002 was deposited on Feb. 6, 1998 with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20108-0971,U.S.A. under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure.

S-RPV-002 was derived from S-RPV-000 (ATCC Strain VR-838). This wasaccomplished utilizing the homology vector 902-19.18 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-galactosidase (BLUOGAL™ ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-RPV-002. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-RPV-002 was assayed for expression of SIV-specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT RPV.Polyclonal swine anti-SIV serum or polyclonal goat anti-HA serum wasshown to react specifically with S-RPV-002 plaques and not withS-RPV-000 negative control plaques. All S-RPV-002 observed plaquesreacted with the antiserum indicating that the virus was stablyexpressing the SIV HA protein.

To confirm the expression of the SIV HA protein gene product, cells wereinfected with S-RPV-002 and samples of infected cell lysates and culturesupernatants were subjected to SDS polyacrylamide gel electrophoresis.The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE.Polyclonal swine anti-SIV serum or polyclonal goat anti-HA serum wasused to detect expression of SIV specific proteins. The cell lysate andculture supernatant from S-RPV-002 infected cells exhibited bandscorresponding to 64 kd, which is the expected size of the SIV HAprotein.

S-RPV-002 is useful as a vaccine in swine against swine influenzainfection. S-RPV-002 is useful as a vaccine in combination withS-RPV-003 which expresses SIV NA. S-RPV-002 is also useful forexpression of the SIV HA protein.

EXAMPLE 4

S-RPV-003

S-RPV-003 is a raccoonpox virus (RPV) that expresses at least twoforeign genes. The gene for E. coli β-galactosidase (lacZ) and the genefor swine influenza virus (SIV) NA (H1N1) were inserted into the RPV O1LORF (a unique NotI restriction site has replaced an XbaI restrictionsite) within the RPV HindIII “U” genomic fragment. The lacZ gene isunder the control of the synthetic late promoter (LP1), and the SIV NAgene is under the control of the synthetic late/early promoter (LP2EP2).

S-RPV-003 was derived from S-RPV-000 (ATCC Strain VR-838). This wasaccomplished utilizing the homology vector 902-49.5 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-galactosidase (BLUOGAL™ ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-RPV-003. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-RPV-003 was assayed for expression of SIV-specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT RPV.Polyclonal swine anti-SIV serum was shown to react specifically withS-RPV-003 plaques and not with S-RPV-000 negative control plaques. AllS-RPV-003 observed plaques reacted with the antiserum indicating thatthe virus was stably expressing the SIV NA protein.

To confirm the expression of the SIV NA protein gene product, cells wereinfected with S-RPV-003 and samples of infected cell lysates and culturesupernatants were subjected to SDS polyacrylamide gel electrophoresis.The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE.Polyclonal goat anti-NA serum was used to detect expression of SIVspecific proteins. The cell lysate and culture supernatant fromS-RPV-003 infected cells exhibited bands corresponding to 64 kd, whichis the expected size of the SIV NA protein.

S-RPV-003 is useful as a vaccine in swine against swine influenzainfection. S-RPV-003 is useful as a vaccine in combination withS-RPV-001 or S-RPV-002 which express SIV HA. S-RPV-003 is also usefulfor expression of the SIV NA protein.

Polyclonal murine antibodies to SIV NA were produced using S-RPV-003according to the PROCEDURE TO PRODUCE MURINE POLYCLONAL ANTIBODIES TOPROTEINS EXPRESSED IN RECOMBINANT RACCOONPOX VIRUS. BLACK PLAQUE ASSAYSwere conducted, using the mouse anti S-RPV-003 serum, againstrecombinant swinepox virus (SPV) expressing swine influenza virus HA orNA proteins. The mouse anti S-RPV-003 serum (1:100 dil) was shown toreact specifically with recombinant SPV expressing NA. The serum did notcross react to either recombinant SPV expressing SIV HA or-galactosidase.

EXAMPLE 5

S-RPV-004

S-RPV-004 is a raccoonpox virus (RPV) that expresses at least twoforeign genes. The gene for E. coli β-galactosidase (lacZ) and the genefor porcine reproductive and respiratory syndrome virus (PRRS) ORF3 wereinserted into the RPV O1L ORF (a unique NotI restriction site hasreplaced an XbaI restriction site) within the RPV HindIII “U” genomicfragment. The lacZ gene is under the control of the synthetic latepromoter (LP1), and the PRRS ORF3 gene is under the control of thesynthetic late/early promoter (LP2EP2).

S-RPV-004 was derived from S-RPV-000 (ATCC Strain VR-838). This wasaccomplished utilizing the homology vector 902-49.14 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-galactosidase (BLUOGAL™ ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-RPV-004. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-RPV-004 was assayed for expression of PRRS-specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT RPV.Polyclonal swine anti-PRRS serum was shown to react specifically withS-RPV-004 plaques and not with S-RPV-000 negative control plaques. AllS-RPV-004 observed plaques reacted with the antiserum indicating thatthe virus was stably expressing the PRRS ORF3 protein.

To confirm the expression of the PRRS ORF3 protein gene product, cellswere infected with S-RPV-004 and samples of infected cell lysates andculture supernatants were subjected to SDS polyacrylamide gelelectrophoresis. The gel was blotted and analyzed using the WESTERNBLOTTING PROCEDURE. A polyclonal swine anti-PRRS serum was used todetect expression of PRRS specific proteins. The cell lysate a ndculture supernatant from S-RPV-004 infected cells exhibited a bandcorresponding to 45 kd, which is the expected size of the PRRS ORF3protein. ORF3 protein is secreted from infected cells into the culturemedia.

S-RPV-004 is useful as a vaccine in swine against PRRS infection.S-RPV-004 is useful as a vaccine in combination with S-RPV-005 andS-RPV-006 which express PRRS ORF4 and PRRS ORF5, respectively. S-RPV-004is also useful for expression of the PRRS ORF3 protein.

Polyclonal murine antibodies to PRRS ORF3 were produced using S-RPV-004according to the PROCEDURE TO PRODUCE MURINE POLYCLONAL ANTIBODIES TOPROTEINS EXPRESSED IN RECOMBINANT RACCOONPOX VIRUS. BLACK PLAQUE ASSAYSare conducted, using the mouse anti S-RPV-004 serum, against recombinantswinepox virus (SPV) expressing PRRS ORF3, ORF4 or ORF5 proteins.

EXAMPLE 6

S-RPV-005

S-RPV-005 is a raccoonpox virus (RPV) that expresses at least twoforeign genes. The gene for E. coli β-galactosidase (lacZ) and the genefor porcine reproductive and respiratory syndrome virus (PRRS) ORF4 wereinserted into the RPV O1L ORF (a unique NotI restriction site hasreplaced an XbaI restriction site) within the RPV HindIII “U” genomicfragment. The lacZ gene is under the control of the synthetic latepromoter (LP1), and the PRRS ORF4 gene is under the control of thesynthetic late/early promoter (LP2EP2).

S-RPV-005 was derived from S-RPV-000 (ATCC Strain VR-838). This wasaccomplished utilizing the homology vector 902-49.23 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-galactosidase (BLOUGAL™ ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-RPV-005. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-RPV-005 was assayed for expression of PRRS-specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT RPV.Polyclonal swine anti-PRRS serum was shown to react specifically withS-RPV-005 plaques and not with S-RPV-000 negative control plaques. AllS-RPV-005 observed plaques reacted with the antiserum indicating thatthe virus was stably expressing the PRRS ORF4 protein.

To confirm the expression of the PRRS ORF4 protein gene product, cellswere infected with S-RPV-005 and samples of infected cell lysates weresubjected to SDS polyacrylamide gel electrophoresis. The gel was blottedand analyzed using the WESTERN BLOTTING PROCEDURE. A polyclonal swineanti-PRRS serum was used to detect expression of PRRS specific proteins.The cell lysate from S-RPV-005 infected cells exhibited a bandcorresponding to 3′ kd, which is the expected size of the PRRS ORF4protein (202 amino acids).

S-RPV-005 is useful as a vaccine in swine against PRRS infection.S-RPV-005 is useful as a vaccine in combination with S-RPV-004 andS-RPV-006 which express PRRS ORF3 and PRRS ORF5, respectively. S-RPV-005is also useful for expression of the PRRS ORF4 protein.

Polyclonal murine antibodies to PRRS ORF4 were produced using S-RPV-005according to the PROCEDURE TO PRODUCE MURINE POLYCLONAL ANTIBODIES TOPROTEINS EXPRESSED IN RECOMBINANT RACCOONPOX VIRUS. BLACK PLAQUE ASSAYSare conducted, using the mouse anti S-RPV-005 serum, against recombinantswinepox virus (SPV) expressing PRRS ORF3, ORF4 or ORF5 proteins.

EXAMPLE 7

S-RPV-006

S-RPV-006 is a raccoonpox virus (RPV) that expresses at least twoforeign genes. The gene for E. coli β-galactosidase (lacZ) and the genefor porcine reproductive and respiratory syndrome virus (PRRS) ORF5 wereinserted into the RPV O1L ORF (a unique NotI restriction site hasreplaced an XbaI restriction site) within the RPV HindIII “U” genomicfragment. The lacZ gene is under the control of the synthetic latepromoter (LP1), and the PRRS ORF5 gene is under the control of thesynthetic late/early promoter (LP2EP2).

S-RPV-006 was derived from S-RPV-000 (ATCC Strain VR-838). This wasaccomplished utilizing the homology vector 902-49.34 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-galactosidase (BLUOGAL™ ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-RPV-006. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

To confirm the expression of the PRRS ORF5 protein gene product, cellswere infected with S-RPV-006 and samples of infected cell lysates weresubjected to SDS polyacrylamide gel electrophoresis. The gel was blottedand analyzed using the WESTERN BLOTTING PROCEDURE. A polyclonal swineanti-PRRS serum was used to detect expression of PRRS specific proteins.The cell lysate from S-RPV-006 infected cells exhibited a bandcorresponding to 26 kd, which is the expected size of the PRRS ORF5protein.

S-RPV-006 is useful as a vaccine in swine against PRRS infection.S-RPV-006 is useful as a vaccine in combination with S-RPV-004 andS-RPV-005 which express PRRS ORF3 and PRRS ORF4, respectively. S-RPV-006is also useful for expression of the PRRS ORF5 protein.

EXAMPLE 8

S-RPV-007

S-RPV-007 is a raccoonpox virus (RPV) that expresses at least oneforeign gene. The gene for E. coli β-galactosidase (lacZ) was insertedinto the RPV O1L ORF (a unique NotI restriction site has replaced anXbaI restriction site) within the RPV HindIII “U” genomic fragment. ThelacZ gene is under the control of the synthetic late promoter (LP1), andsecond foreign DNA is inserted into a unique EcoRI or BamHI site underthe control of the synthetic late/early promoter (LP2EP2).

S-RPV-007 was derived from S-RPV-000 (ATCC Strain VR-838). This wasaccomplished utilizing the homology vector 902-49.46 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-galactosidase (BLOUGAL™ ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-RPV-007. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-RPV-007 is useful as a vaccine in mammalian and avian species. When aforeign DNA encoding an antigenic polypeptide form an avian or mammalianpathogen, or encoding a cytokine or immune-modulating protein isinserted under the control of the LP2EP2 promoter in an EcoRI or BamHIsite in S-RPV-007, the resulting recombinant virus is useful as avaccine against the avian or mammalian pathogen.

The XbaI site within the RPV genomic fragment HindIII U is a usefulinsertion site for foreign DNA. A second foreign DNA is inserted intoS-RPV-007 at a unique EcoRI or BamHI insertion site and is under controlof the synthetic late/early promoter, LP2EP2. When the second foreignDNA encodes an antigenic polypeptide from an avian or mammalianpathogen, or encodes a cytokine or immune-modulating protein, theresulting recombinant virus is useful as a vaccine against the avian ormammalian pathogen.

EXAMPLE 9

S-RPV-008

S-RPV-008 (ATTC Accession No. VR-2598) is a raccoonpox virus (RPV) thatexpresses at least one foreign gene. The gene for E. coliβ-galactosidase (lacZ) was inserted into a unique SnaB I site within theRPV genomic fragment HindIII N (a unique NotI restriction site hasreplaced a SnaBI restriction site). The lacZ gene is under the controlof the synthetic late promoter (LP1), and second foreign DNA is insertedinto a unique EcoRI or BamHI site under the control of the syntheticlate/early promoter (LP2EP2).

S-RPV-008 was deposited on February 6, 1998 with the American TypeCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va20108-0971, U.S.A. under the provisions of the Budapest Treaty for theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure.

S-RPV-008 was derived from S-RPV-000 (ATCC Strain VR-838). This wasaccomplished utilizing the homology vector 902-67.1 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-galactosidase (BLUOGAL™ ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-RPV-008. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-RPV-008 is useful as a vaccine in mammalian and avian species. When aforeign DNA encoding an antigenic polypeptide form an avian or mammalianpathogen, or encoding a cytokine or immune-modulating protein isinserted under the control of the LP2EP2 promoter in an EcoRI or BamHIsite in S-RPV-008, the resulting recombinant virus is useful as avaccine against the avian or mammalian pathogen.

The SnaBI site within the RPV genomic fragment HindIII N is a usefulinsertion site for foreign DNA. A second foreign DNA is inserted intoS-RPV-008 at a unique EcoRI or BamHI insertion site and is under controlof the synthetic late/early promoter, LP2EP2. When the second foreignDNA encodes an antigenic polypeptide from an avian or mammalianpathogen, or encodes a cytokine or immune-modulating protein, theresulting recombinant virus is useful as a vaccine against the avian ormammalian pathogen.

EXAMPLE 10

S-RPV-009

S-RPV-009 (ATCC Accession No. VR-2599) is a raccoonpox virus (RPV) thatexpresses at least one foreign gene. The gene for E. coliβ-galactosidase (lacZ) was inserted into a unique EcoRV site within theRPV genomic fragment HindIII N (a unique NotI restriction site hasreplaced an EcoRV restriction site). The lacZ gene is under the controlof the synthetic late promoter (LP1).

S-RPV-009 was deposited on Feb. 6, 1998 with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20108-0971,U.S.A. under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure.

S-RPV-009 was derived from S-RPV-000 (ATCC Strain VR-838). This wasaccomplished utilizing the homology vector 902-67.14 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING 1-galactosidase (BLOUGAL™ ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-RPV-009. This virus was assayed for1-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-RPV-009 is useful as a vaccine in mammalian and avian species. When aforeign DNA encoding an antigenic polypeptide form an avian or mammalianpathogen, or encoding a cytokine or immune-modulating protein isinserted under the control of the LP2EP2 promoter in an EcoRI or BamHIsite in S-RPV-009, the resulting recombinant virus is useful as avaccine against the avian or mammalian pathogen.

The EcoRV site within the RPV genomic fragment HindIII N is a usefulinsertion site for foreign DNA. A second foreign DNA is inserted intoS-RPV-009 at a unique EcoRI or BamHI insertion site and is under controlof the synthetic late/early promoter, LP2EP2. When the second foreignDNA encodes an antigenic polypeptide from an avian or mammalianpathogen, or encodes a cytokine or immune-modulating protein, theresulting recombinant virus is useful as a vaccine against the avian ormammalian pathogen.

EXAMPLE 11

S-RPV-010

S-RPV-010 is a raccoonpox virus (RPV) that expresses at least oneforeign gene. The gene for E. coli β-galactosidase (lacZ) was insertedinto a unique HpaI site within the RPV genomic fragment HindIII M (aunique NotI restriction site has replaced a HpaI restriction site). ThelacZ gene is under the control of the synthetic late promoter (LP1).

S-RPV-010 was derived from S-RPV-000 (ATCC Strain VR-838). This wasaccomplished utilizing the homology vector 902-67.27(see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-galactosidase (BLOUGAL™ ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-RPV-010. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods.

S-RPV-010 is useful as a vaccine in mammalian and avian species. When aforeign DNA encoding an antigenic polypeptide form an avian or mammalianpathogen, or encoding a cytokine or immune-modulating protein isinserted under the control of the LP2EP2 promoter in an EcoRI or BamHIsite in S-RPV-010, the resulting recombinant virus is useful as avaccine against the avian or mammalian pathogen.

The HpaI site within the RPV genomic fragment HindIII M is a usefulinsertion site for foreign DNA. A second foreign DNA is inserted intoS-RPV-010 at a unique EcoRI or BamHI insertion site and is under controlof the synthetic late/early promoter, LP2EP2. When the second foreignDNA encodes an antigenic polypeptide from an avian or mammalianpathogen, or encodes a cytokine or immune-modulating protein, theresulting recombinant virus is useful as a vaccine against the avian ormammalian pathogen.

EXAMPLE 12

S-RPV-011

S-RPV-011 is a raccoonpox virus (RPV) that expresses at least oneforeign gene. The gene for E. coli β-glucuronidase (uidA) was insertedinto the RPV O1L ORF (a unique NotI restriction site has replaced anXbaI restriction site) within the RPV HindIII “U” genomic fragment. Theuida gene is under the control of the synthetic late promoter (LP1).

S-RPV-011 was derived from S-RPV-000 (ATCC Strain VR-838). This wasaccomplished utilizing the homology vector 919-16.12 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-glucuronidase (X-GLUC™ASSAYS). The final result of blue plaque purification was therecombinant virus designated S-RPV-011. This virus was assayed forβ-glucuronidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-RPV-011 is useful as a vaccine in mammalian and avian species. When asecond foreign DNA encoding an antigenic polypeptide from an avian ormammalian pathogen, or encoding a cytokine or immune-modulating proteinis inserted under the control of the LP2EP2 promoter in a unique EcoRIor BamHI site in S-RPV-011, the resulting recombinant virus is useful asa vaccine against the avian or mammalian pathogen.

EXAMPLE 13

S-RPV-012

S-RPV-012 is a raccoonpox virus (RPV) that expresses at least twoforeign genes. The gene for E. coli β-galactosidase (lacZ) and the genefor porcine parvovirus (PPV) VP2 capsid protein were inserted into theRPV O1L ORF (a unique NotI restriction site has replaced an XbaIrestriction site) within the RPV HindIII “U” genomic fragment. The lacZgene is under the control of the synthetic late promoter (LP1), and thePPV VP2 gene is under the control of the synthetic late/early promoter(LP2EP2).

S-RPV-012 was derived from S-RPV-000 (ATCC Strain VR-838). This wasaccomplished utilizing the homology vector 919-63.21 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-galactosidase (BLOUGAL™ ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-RPV-012. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-RPV-012 is useful as a vaccine in swine against porcine parvovirusinfection. S-RPV-012 is also useful for expression of the PPV VP2protein.

EXAMPLE 14

S-RPV-013

S-RPV-013 is a raccoonpox virus (RPV) that expresses at least oneforeign gene. The gene for E. coli β-glucuronidase (uidA) is insertedinto the RPV O1L ORF (a unique NotI restriction site has replaced anXbaI restriction site) within the RPV HindIII “U” genomic fragment. Theuida gene is under the control of the synthetic late promoter (LP1).

S-RPV-013 is derived from S-RPV-000 (ATCC Strain VR-838). This isaccomplished utilizing the HOMOLOGY VECTOR 935-50.1 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock is screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-glucuronidase (X-GLUC™ASSAYS). The final result of blue plaque purification is the recombinantvirus designated S-RPV-013. This virus is assayed for β-glucuronidaseexpression, purity, and insert stability by multiple passages monitoredby the blue plaque assay as described in Materials and Methods. Afterthe initial three rounds of purification, all plaques observed are blueindicating that the virus is pure, stable, and expressing the foreigngene.

S-RPV-013 is useful as a vaccine in mammalian and avian species. When asecond foreign DNA encoding an antigenic polypeptide from an avian ormammalian pathogen, or encoding a cytokine or immune-modulating proteinis inserted under the control of a promoter in a unique site inS-RPV-013, the resulting recombinant virus is useful as a vaccineagainst the avian or mammalian pathogen.

EXAMPLE 15

S-RPV-014

S-RPV-014 is a raccoonpox virus (RPV) that expresses at least oneforeign gene. The gene for E. coli β-galactosidase (lacZ) is insertedinto the RPV O1L ORF (a unique NotI restriction site has replaced anXbaI restriction site) within the RPV HindIII “U” genomic fragment. ThelacZ gene is under the control of the swinepox virus I5L promoter (seeFIG. 9).

S-RPV-014 is derived from S-RPV-000 (ATCC Strain VR-838). This isaccomplished utilizing the HOMOLOGY VECTOR FOR S-RPV-014 (see Materialsand Methods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT RPV. The transfection stock isscreened by the SCREEN FOR RECOMBINANT RPV EXPRESSING β-GALACTOSIDASE(BLUOGAL ASSAY). The final result of blue plaque purification is therecombinant virus designated S-RPV-014. This virus is assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved are blue indicating that the virus is expressing the foreigngene.

S-RPV-014 is useful as a vaccine in mammalian and avian species. When asecond foreign DNA encoding an antigenic polypeptide from an avian ormammalian pathogen, or encoding a cytokine or immune-modulating proteinis inserted under the control of a promoter in a unique site inS-RPV-014, the resulting recombinant virus is useful as a vaccineagainst the avian or mammalian pathogen. The SPV I5L promoter is auseful heterologous promoter in a recombinant raccoonpox virus. THE SPVI5L promoter is a strong late promoter.

EXAMPLE 16

S-RPV-015

S-RPV-015 is a raccoonpox virus (RPV) that expresses at least twoforeign genes. The gene for E. coli β-glucuronidase (uidA) and the genefor porcine reproductive and respiratory syndrome virus (PRRS) ORF2(Eichelberger strain) are inserted into the RPV O1L ORF (a unique NotIrestriction site has replaced an XbaI restriction site) within the RPVHindIII “U” genomic fragment. The uida gene is under the control of thesynthetic late promoter (LP1), and the PRRS ORF2 gene is under thecontrol of the synthetic early promoter (EP2).

S-RPV-015 is derived from S-RPV-000 (ATCC Strain VR-838). This isaccomplished utilizing the HOMOLOGY VECTOR 934-65.1 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock is screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-glucuronidase (X-GLUC™ASSAYS). The final result of blue plaque purification is the recombinantvirus designated S-RPV-015. This virus is assayed for β-glucuronidaseexpression, purity, and insert stability by multiple passages monitoredby the blue plaque assay as described in Materials and Methods. Afterthe initial three rounds of purification, all plaques observed were blueindicating that the virus is pure, stable, and expressing the foreigngene.

S-RPV-015 is assayed for expression of PRRS-specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT RPV.Rabbit polyclonal anti β-glucuronidase antibody is shown to reactspecifically with S-RPV-015 plaques and not with S-RPV-000 negativecontrol plaques.

S-RPV-015 is useful as a vaccine in swine against PRRS infection.S-RPV-015 is useful as a vaccine in combination with S-RPV-016,S-RPV-017, S-RPV-018, S-RPV-019, and S-RPV-020 which express PRRS ORF3,ORF4, ORF5, ORF6 and ORF7, respectively. S-RPV-015 is also useful forexpression of the PRRS ORF2 protein for purposes of raising antibodiesto PRRS ORF2 and for diagnostic assays.

EXAMPLE 17

S-RPV-016

S-RPV-016 is a raccoonpox virus (RPV) that expresses at least twoforeign genes. The gene for E. coli β-glucuronidase (uidA) and the genefor porcine reproductive and respiratory syndrome virus (PRRS) ORF3(Eichelberger strain) are inserted into the RPV O1L ORF (a unique NotIrestriction site has replaced an XbaI restriction site) within the RPVHindIII “U” genomic fragment. The uida gene is under the control of thesynthetic late promoter (LP1), and the PRRS ORF3 gene is under thecontrol of the synthetic late/early promoter (LP2EP2).

S-RPV-016 is derived from S-RPV-000 (ATCC Strain VR-838). This isaccomplished utilizing the HOMOLOGY VECTOR 934-64.2 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock is screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-glucuronidase (X-GLUC™ASSAYS). The final result of blue plaque purification is the recombinantvirus designated S-RPV-016. This virus is assayed for β-glucuronidaseexpression, purity, and insert stability by multiple passages monitoredby the blue plaque assay as described in Materials and Methods. Afterthe initial three rounds of purification, all plaques observed were blueindicating that the virus is pure, stable, and expressing the foreigngene.

S-RPV-016 is assayed for expression of PRRS-specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECCMBINANT RPV.Rabbit polyclonal anti β-glucuronidase antibody is shown to reactspecifically with S-RPV-016 plaques and not with S-RPV-000 negativecontrol plaques.

S-RPV-016 is useful as a vaccine in swine against PRRS infection.S-RPV-016 is useful as a vaccine in combination with S-RPV-015,S-RPV-017, S-RPV-018, S-RPV-019, and S-RPV-020 which express PRRS ORF2,ORF4, ORF5, ORF6 and ORF7, respectively. S-RPV-016 is also useful forexpression of the PRRS ORF3 protein for purposes of raising antibodiesto PRRS ORF3 and for diagnostic assays.

EXAMPLE 18

S-RPV-017

S-RPV-017 is a raccoonpox virus (RPV) that expresses at least twoforeign genes. The gene for E. coli β-glucuronidase (uidA) and the genefor porcine reproductive and respiratory syndrome virus (PRRS) ORF4(Eichelberger strain) are inserted into the RPV O1L ORF (a unique NotIrestriction site has replaced an XbaI restriction site) within the RPVHindIII “U” genomic fragment. The uidA gene is under the control of thesynthetic late promoter (LP1), and the PRRS ORF4 gene is under thecontrol of the synthetic late/early promoter (LP2EP2).

S-RPV-017 is derived from S-RPV-000 (ATCC Strain VR-838). This isaccomplished utilizing the HOMOLOGY VECTOR 938-94.1 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock is screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-glucuronidase (X-GLUC™ASSAYS). The final result of blue plaque purification is the recombinantvirus designated S-RPV-017. This virus is assayed for β-glucuronidaseexpression, purity, and insert stability by multiple passages monitoredby the blue plaque assay as described in Materials and Methods. Afterthe initial three rounds of purification, all plaques observed were blueindicating that the virus is pure, stable, and expressing the foreigngene.

S-RPV-017 is assayed for expression of PRRS-specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT RPV.Rabbit polyclonal anti β-glucuronidase antibody is shown to reactspecifically with S-RPV-017 plaques and not with S-RPV-000 negativecontrol plaques.

S-RPV-017 is useful as a vaccine in swine against PRRS infection.S-RPV-017 is useful as a vaccine in combination with S-RPV-015,S-RPV-016, S-RPV-018, S-RPV-019, and S-RPV-020 which express PRRS ORF2,ORF3, ORF5, ORF6 and ORF7, respectively. S-RPV-017 is also useful forexpression of the PRRS ORF4 protein for purposes of raising antibodiesto PRRS ORF4 and for diagnostic assays.

EXAMPLE 19

S-RPV-018

S-RPV-018 is a raccoonpox virus (RPV) that expresses at least twoforeign genes. The gene for E. coli β-glucuronidase (uidA) and the genefor porcine reproductive and respiratory syndrome virus (PRRS) ORF5(Eichelberger strain) are inserted into the RPV O1L ORF (a unique NotIrestriction site has replaced an XbaI restriction site) within the RPVHindIII “U” genomic fragment. The uidA gene is under the control of thesynthetic late promoter (LP1), and the PRRS ORF5 gene is under thecontrol of the synthetic early promoter (EP2).

S-RPV-018 is derived from S-RPV-000 (ATCC Strain VR-838). This isaccomplished utilizing the HOMOLOGY VECTOR 938-94.25 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock is screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-glucuronidase (X-GLUC™ASSAYS). The final result of blue plaque purification is the recombinantvirus designated S-RPV-018. This virus is assayed for β-glucuronidaseexpression, purity, and insert stability by multiple passages monitoredby the blue plaque assay as described in Materials and Methods. Afterthe initial three rounds of purification, all plaques observed were blueindicating that the virus is pure, stable, and expressing the foreigngene.

S-RPV-018 is assayed for expression of PRRS-specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT RPV.Rabbit polyclonal anti β-glucuronidase antibody is shown to reactspecifically with S-RPV-018 plaques and not with S-RPV-000 negativecontrol plaques.

S-RPV-018 is useful as a vaccine in swine against PRRS infection.S-RPV-018 is useful as a vaccine in combination with S-RPV-015,S-RPV-016, S-RPV-017, S-RPV-019, and S-RPV-020 which express PRRS ORF2,ORF3, ORF4, ORF6 and ORF7, respectively. S-RPV-018 is also useful forexpression of the PRRS ORF5 protein for purposes of raising antibodiesto PRRS ORF5 and for diagnostic assays.

EXAMPLE 20

S-RPV-019

S-RPV-019 is a raccoonpox virus (RPV) that expresses at least twoforeign genes. The gene for E. coli β-glucuronidase (uidA) and the genefor porcine reproductive and respiratory syndrome virus (PRRS) ORF6(Eichelberger strain) are inserted into the RPV O1L ORF (a unique NotIrestriction site has replaced an XbaI restriction site) within the RPVHindIII “U” genomic fragment. The uidA gene is under the control of thesynthetic late promoter (LP1), and the PRRS ORF6 gene is under thecontrol of the synthetic late promoter (LP2).

S-RPV-019 is derived from S-RPV-000 (ATCC Strain VR-838). This isaccomplished utilizing the HOMOLOGY VECTOR 950-13.4 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock is screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-glucuronidase (X-GLUC™ASSAYS). The final result of blue plaque purification is the recombinantvirus designated S-RPV-019. This virus is assayed for β-glucuronidaseexpression, purity, and insert stability by multiple passages monitoredby the blue plaque assay as described in Materials and Methods. Afterthe initial three rounds of purification, all plaques observed are blueindicating that the virus is expressing the foreign gene.

S-RPV-019 is useful as a vaccine in swine against PRRS infection.S-RPV-019 is useful as a vaccine in combination with S-RPV-015,S-RPV-016, S-RPV-017, S-RPV-018, and S-RPV-020 which express PRRS ORF2,ORF3, ORF4, ORF5 and ORF7, respectively. S-RPV-019 is also useful forexpression of the PRRS ORF6 protein for purposes of raising antibodiesto PRRS ORF6 and for diagnostic assays.

EXAMPLE 21

S-RPV-020

S-RPV-020 is a raccoonpox virus (RPV) that expresses at least twoforeign genes. The gene for E. coli β-glucuronidase (uidA) and the genefor porcine reproductive and respiratory syndrome virus (PRRS) ORF7(Eichelberger strain) are inserted into the RPV O1L ORF (a unique NotIrestriction site has replaced an XbaI restriction site) within the RPVHindIII “U” genomic fragment. The uida gene is under the control of thesynthetic late promoter (LP1), and the PRRS ORF7 gene is under thecontrol of the synthetic early promoter (EP2).

S-RPV-020 is derived from S-RPV-000 (ATCC Strain VR-838). This isaccomplished utilizing the HOMOLOGY VECTOR 936-87.2 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock is screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-glucuronidase (X-GLUC™ASSAYS). The final result of blue plaque purification is the recombinantvirus designated S-RPV-020. This virus is assayed for β-glucuronidaseexpression, purity, and insert stability by multiple passages monitoredby the blue plaque assay as described in Materials and Methods.

After the initial three rounds of purification, all plaques observedwere blue indicating that the virus is pure, stable, and expressing theforeign gene.

S-RPV-020 is assayed for expression of PRRS-specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT RPV.Rabbit polyclonal anti β-glucuronidase antibody is shown to reactspecifically with S-RPV-020 plaques and not with S-RPV-000 negativecontrol plaques.

S-RPV-020 is useful as a vaccine in swine against PRRS infection.S-RPV-020 is useful as a vaccine in combination with S-RPV-015,S-RPV-016, S-RPV-017, S-RPV-018, and S-RPV-019 which express PRRS ORF2,ORF3, ORF4, ORF5 and ORF6, respectively. S-RPV-020 is also useful forexpression of the PRRS ORF7 protein for purposes of raising antibodiesto PRRS ORF7 and for diagnostic assays.

EXAMPLE 22

S-RPV-028

S-RPV-028 is a raccoonpox virus (RPV) that expresses at least threeforeign genes. The gene for E. coli β-glucuronidase (uidA) and the genesfor porcine reproductive and respiratory syndrome virus (PRRS) ORF5 andORF6 (Eichelberger strain) are inserted into the RPV O1L ORF (a uniqueNotI restriction site has replaced an XbaI restriction site) within theRPV HindIII “U” genomic fragment. The uida gene is under the control ofthe synthetic late promoter (LP1), the PRRS ORF5 gene is under thecontrol of the synthetic early promoter (EP2) and the PRRS ORF6 gene isunder the control of the synthetic late promoter (LP1).

S-RPV-028 is derived from S-RPV-000 (ATCC Strain VR-838). This isaccomplished utilizing the HOMOLOGY VECTOR 950-16.34 (see Materials andMethods) and virus S-RPV-000 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT RPV. The transfection stock is screened bythe SCREEN FOR RECOMBINANT RPV EXPRESSING β-glucuronidase (X-GLUC™ASSAYS). The final result of blue plaque purification is the recombinantvirus designated S-RPV-028. This virus is assayed for β-glucuronidaseexpression, purity, and insert stability by multiple passages monitoredby the blue plaque assay as described in Materials and Methods. Afterthe initial three rounds of purification, all plaques observed were blueindicating that the virus is pure, stable, and expressing the foreigngene.

S-RPV-028 is assayed for expression of PRRS-specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT RPV.Rabbit polyclonal anti β-glucuronidase antibody is shown to reactspecifically with S-RPV-020 plaques and not with S-RPV-000 negativecontrol plaques.

S-RPV-028 is useful as a vaccine in swine against PRRS infection.S-RPV-028 is useful as a vaccine in combination with S-RPV-015,S-RPV-016, S-RPV-017, which express PRRS ORF2, ORF3, and ORF4,respectively. S-RPV-028 is also useful for expression of the PRRS ORF5and ORF6 proteins for purposes of raising antibodies to PRRS ORF5 andORF6 and for diagnostic assays.

EXAMPLE 23

Expression of the RPV K1L or C7L Genes in Recombinant Swinepox Virus toBroaden the Host Range of Recombinant Swinepox Virus

The K1L gene in vaccinia virus determines the range of replication ofthe virus in the animal host. Deletion of this gene in vaccinia virusrestricts viral replication in the host animal. Sequencing of the nearterminal region of the swinepox virus genome indicates that the K1L geneis not present in this region and may be absent in the swinepox virusgenome. (26). The K1L homologue in raccoonpox virus is located withinthe HindIII “T” RPV genomic fragment (SEQ ID NO. 5 and 10). The hostrange of raccoonpox is broader than swinepox virus (which replicatesmainly in swine); Raccoonpox host range includes raccoons, dogs, cats,swine, cattle, monkeys and other mammals (See Table 1). A recombinantswinepox virus comprising the RPV K1L ORF under the control of thesynthetic pox promoter, K1L promoter or LP2EP2 (or LP1 or LP2 or EP1 orEP2), has a broader host range to include raccoons, dogs, cats, andother mammals. Preferably, the promoter is the K1L promoter fromraccoonpox which will express the K1L protein at the correct stage ofthe viral life cycle. A modified host range recombinant swinepox virus,which also expresses foreign DNA coding for an antigen from a diseasepathogen, is an improved vaccine in raccoons, dogs, cats, and othermammals since it will replicate in the animals, expressing the foreignDNA and elicit a strong immune response against the antigen from thedisease pathogen. The modified host range recombinant swinepox virusdoes not cause disease in the vaccinated animal.

In an alternative embodiment, recombinant swinepox virus comprising theRPV C7L ORF (host range gene) under the control of the synthetic poxpromoter, C7L promoter or LP2EP2 (or LP1 or LP2 or EP1 or EP2), has abroader host range to include raccoons, dogs, cats, and other mammals.Preferably, the promoter is the C7L promoter from raccoonpox which willexpress the C7L protein at the correct stage of the viral life cycle. Amodified host range recombinant swinepox virus, which also expressesforeign DNA coding for an antigen from a disease pathogen, is animproved vaccine in raccoons, dogs, cats, and other mammals since itwill replicate in the animals, expressing the foreign DNA and elicit astrong immune response against the antigen from the disease pathogen.The modified host range recombinant swinepox virus does not causedisease in the vaccinated animal.

S-SPV-248:

S-SPV-248 is a swinepox virus (SPV) that expresses at least two foreigngenes. The gene for raccoonpox virus K1L host range is inserted into aunique NotI restriction site located distal to the E. Coliβ-galactosidase (lacZ) gene contained in a plasmid homology vector whichdeletes the swinepox virus I4L and I5L genes. The lacZ gene is under thecontrol of the swinepox I5L and the K1L gene is under control of theraccoonpox virus K1L promoter.

SPV-248 is derived from S-SPV-001 (Kasza Strain). This was accomplishedutilizing a homology vector and virus S-SPV-001 in the HOMOLOGOUSRECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. The transfectionstock is screened using the SCREEN FOR RECOMBINANT SPV EXPRESSINGβ-galactosidase (SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATICMARKER GENES). The final result of blue plaque purification is therecombinant virus designated S-SPV-248. This virus is assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods.

S-SPV-248 is useful as a vaccine against disease in mammals. S-SPV-248has a broader host range than other recombinant SPV due to expression ofthe RPV K1L gene. The host range includes, but is not limited to,raccoons, dogs, cats, horses, cows, sheep, goats, and other mammals.S-SPV-248 is also useful as a vaccine in avian species.

S-SPV-249:

S-SPV-249 is a swinepox virus (SPV) that expresses at least two foreigngenes. The gene for raccoonpox virus C7L host range is inserted into aunique NotI restriction site located distal to the E. Coliβ-galactosidase (lacZ) gene contained in a plasmid homology vector whichdeletes the swinepox virus I4L and I5L genes. The lacZ gene is under thecontrol of the swinepox I5L promoter and the C7L gene is under thecontrol of the raccoonpox virus C7L promoter.

SPV-249 is derived from S-SPV-001 (Kasza Strain). This was accomplishedutilizing a homology vector and virus S-SPV-001 in the HOMOLOGOUSRECOMBINATION PROCEDURE FOR GENERATING SPV. The transfection stock isscreened using the SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase(SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES).The final result of blue plaque purification is the recombinant virusdesignated S-SPV-249. This virus is assayed for β-galactosidaseexpression, purity, and insert stability by multiple passages monitoredby the blue plaque assay as described in Material and Methods.

S-SPV-249 is useful as a vaccine against disease in mammals. S-SPV-249has a broader host range than other recombinant SPV due to expression ofthe RPV C7L gene. The host range includes, but is not limited to,raccoons, dogs, cats, horses, cows, sheep, goats and other mammals.S-SPV-249 is also useful as a vaccine in avian species.

Deletion of Host Range Genes from Recombinant Raccoonpox Virus:

In a further embodiment, deletion of the host range genes selected fromthe group consisting of C7L, C6L, C5L, C4L, C3L, C2L, C1L, N1L, N2L,M1L, M2L, and K1L (“C7L to K1L”) in a raccoonpox virus vector results inlimited replication of the recombinant raccoonpox in Vero cells and noreplication in other cell types, such as CRFK or MDCK cells. Therecombinant raccoonpox with a deletion of some or all of the C7L to K1Lhost range genes is useful as a vaccine which is safe and less virulentthan recombinant raccoonpox containing the C7L to K1L genes. To selectfor recombinant raccoonpox expressing a foreign DNA of interest, ahomology vector containing some or all of the host range genes C7L toK1L and a foreign gene of interest under the control of a promoter iscombined with recombinant raccoonpox virus deleted for the C7L to K1Lgenes in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT RPV and selected for growth in cell types other than Verocells, such as CRFK or MDCK cells. Any recombinant virus which grows inthe other cell types must contain the host range genes C7L to K1L andthe foreign gene of interest.

TABLE 1 ESK-4 PKWRL VERO CRFK MDCK MDBK Virus (Porcine) (Porcine)(Monkey) (Feline) (Canine) (Bovine) Raccoon + + +++ +++ +++ +/− poxvirus Swinepox virus +++ +++ +++ − − − Vaccinia virus +++ ND +++ +++ ++++++ Swinepox virus +++ +++ +++ +++ +++ +/− with RcnPV K1L gene +++Extensive CPE − No CPE ND Not Determined

EXAMPLE 24

Nonessential Regions in RPV Genomic Fragments W, T, and P for Insertionof Foreign DNA into a Recombinant Raccoonpox Virus.

Nonessential insertion regions in RPV genomic fragments W, T, and P areuseful for constructing recombinant raccoonpox viruses expressingforeign DNA which is inserted into a genomic region of the recombinantRPV. RPV genomic fragments W, T, and P contain open reading frames forvaccinia virus homologues N1L, N2L, M1L, M2L, K1L, K2L, and K7R. (SEQ IDNO. 5-13) (17) These are all nonessential genes and are useful forinsertion of foreign DNA to produce a recombinant raccoonpox virus.Intergenic regions between the ORFs are also useful for insertion offoreign DNA. Some of the restriction sites which are useful forinsertion of foreign DNA in this region are shown in FIG. 2. Preferredinsertion sites for foreign DNA in the HindIII W genomic fragment areBcl I, Acc I, Pvu II, and Xba I; Preferred insertion sites for foreignDNA in the HindIII T genomic fragment are EcoR I, BamH I, Sal I, Bgl II,Pst I, Acc I, Pvu II, Xho I, and Xba I; Preferred insertion sites forforeign DNA in the HindIII P genomic fragment are Bcl I, Acc I, Xmn Iand Xba I.

N1L is a virulence gene (virokine); K1L is a host range gene. It isimportant for use in swinepox virus vector to broaden the host range ofSPV; K2L is a serine protease inhibitor (SPI-3); K3L is a translationinitiation factor; confers interferon resistance; K4L is a member of thephospholipase D superfamily.

EXAMPLE 25

Nonessential Regions in RPV Genomic Fragment S for Insertion of ForeignDNA into a Recombinant Raccoonpox Virus.

Nonessential insertion regions in RPV genomic fragment S are useful forconstructing recombinant raccoonpox viruses expressing foreign DNA whichis inserted into a genomic region of the recombinant RPV. RPV genomicfragment S contains open reading frames for vaccinia virus homologuesB19R, B20R and variola B18R. (SEQ ID NO. 33 to 35) (27). Amino acids 7to 523 of SEQ ID NO 35 has homology to variola virus B18R ORF. Aminoacids 235 to 399 has homology to vaccinia virus B20R ORF. These are allnonessential genes and are useful for insertion of foreign DNA toproduce a recombinant raccoonpox virus. Intergenic regions between theORFs are also useful for insertion of foreign DNA. Some of therestriction sites which are useful for insertion of foreign DNA in thisregion are shown in FIG. 8. Preferred insertion sites for foreign DNA inthe HindIII S genomic fragment are Bgl II, EcoR I, Pme I, Spe I, Sal I,Acc I, BamH I, Bcl I, and Nde I.

Vaccinia B19R is a surface antigen present on infected cells. VacciniaB20R and Variola B18R are homologous to neuraxin protein and map 1Bprotein (25).

EXAMPLE 26

Mapping of Raccoonpox Virus Genomic Fragments HindIII M and HindIII R

Restriction endonuclease mapping and DNA sequencing of the raccoonpoxvirus genome indicates differences in the HindIII restriction mapcompared to the published HindIII restriction map. In the presentinvention, the size of the HindIII M genomic region of RPV is 6.0 kb.The previously published size of the HindIII M genomic region of RPV is5.4 kb (5). In the present invention, the HindIII J and HindIII Wgenomic fragments of RPV are immediately adjacent. An earlier publishedHindIII restriction map of the RPV genome shows HindIII R genomicfragment between HindIII J and HindIII W genomic fragments (6).

EXAMPLE 27

Raccoonpox Virus E11L Promoter

The raccoonpox virus E11L promoter is useful as a homologous promoter inraccoonpox or a heterologous promoter in other poxviruses for theexpression of foreign DNA in a recombinant poxvirus (FIG. 7; SEQ ID NO:30). Other poxviruses include, but are not limited to, swinepox,fowlpox, avipox, canarypox, cowpox and vaccinia virus.

EXAMPLE 28

Swinepox Virus I5L Promoter

The swinepox virus (SPV) I5L promoter is useful as a homologous promoterin swinepox or a heterologous promoter in other poxviruses for theexpression of foreign DNA in a recombinant poxvirus (FIG. 9; SEQ ID NO:55). Other poxviruses include, but are not limited to, raccoonpox,fowlpox, avipox, canarypox, cowpox and vaccinia virus. The SPV I5Lpromoter is contained within the SPV HindIII N genomic fragment. The SPVI5L promoter is a strong late promoter.

References

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27. Perkus, et al., (1991) Virology 180, 406-410

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 58(2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 2061 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM:Raccoonpox virus ATCC# VR-838 (ix) FEATURE: (A) NAME/KEY: CDS (B)LOCATION: 386..1 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 2061..372(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AAGCTTAATC CATCATAAAA TTTATGGTTAGTAACGACTG AAATAACTGC GTAATCATTT 60 TTTGGTACAA TTCTAGTCGG CATAGATTCTGTAACAATGA ATTCGATTCC CGTTACTTTA 120 GTAAAATCTT TTACAAATAA CAAGGATTCATCAAATACGT AAAACTCATT ATTTACTATA 180 GAAATTGATC CCCTATCACA CTTGAAATAAAAAATATCTT TATCCTTTAA CACCAAATAA 240 AATTCAGATT GGTCAATATG AATGTATTCACTTAGTAATT CAACGAACTT ATTTATTAAT 300 TCTGATACAC ACACTGATTC TGTATTGTTTATCGAAAACT TTACTCTTCC TGTATCAGTT 360 TCTAAAAAAA TATTAACAAG TTCCATTTATATCATCCAAT ATTATTGAAA TGGTGGTAAT 420 AGACAAATGA TATAGGTAAG ACGGCACAGTACCTTTGTCG ACCATCTCCT CCATTTCATG 480 CTCTATCTTA TCATGAACTT TGATATGTGAAAACAATACA CCACATGCTT CTATAACAGT 540 ATGTAACACT TTAGATACAA AATGCTTGATACCATCGTAA TTGTTCAACA CGGCCAATCT 600 ATAATAGATA GTAGCTACTA TATATTCTATGATAGTATTG AAGAAGATAA CCACCTTGGC 660 ATATTGGTCA TTTAATACAG ACATGGTATCAACAGATAGC TTAAACGATA GGGAATCCGT 720 GAATGGAATA AGCGTTTCAT TGATTGAATATCCATATACT AACATATCGG ATATCCTGAT 780 GTGTTCCATT AAATCGTTAA GTTTTTTCTTTTTAACCTCG TTGAACATCA TTTCTGTTAA 840 CGGTCCCCAA CATCTTTGAC CAATTAATTTTTGATTTATT TTTCCATGTA TGGCGTATCT 900 AGTCAGGTCG TATAACCTAT CCAATAATCCATCATCGGTA TCCAAATTAC ATACTATGCT 960 TTTCAATTCT CTATAGAAAA GATTAATACATCTAGAACAA CAACATGATG TAATATCTTT 1020 ATCATCAACA GATGTGATAT ATTTGAAGATTTTTCTATGA TAATGAAGAA TTTTTGAGAA 1080 TACTGTATTA ATGGTGTCTG TTACCATGATCCCTTTGATT GCTGACAGAG TCAGCGAGCA 1140 TGATTTCCAA TCTTTAACAA TTTTTATTACCATTATCTTT GTTTTAATGT CTATAGATGA 1200 TAACATAACA CGTCTAACAA TACACGGATTAAGACGGAAA GATGAAATTA TTCTTTCAAC 1260 ATCTCCAATG GATAACTTAC TATTTCGACGTGCATTGTCT ATATGCTCGA GAACATCCTC 1320 CAACGAGTCT GTATCTTTTT TGATTATCGTCGATCTCAAC GATTTGGGTC GTCTAGATCG 1380 TCGGACTCTA TCACCAGATG GCATAGTTATTAGTCTTCTT TCTGTTTTCA TAATGAAATT 1440 TCTAAATTCA TCTGCGAATC TTCTATATCTAAAATCATAA TATGCGATAT TTGTTTCAAC 1500 AAATTTCCGT TCGTCCAATG TCAACATATCTATCTCGGTT TCATACCCTA AATTGAACAT 1560 GGCTACGGAT TTAATTTTAT ATTCATCTATCAACTCTTCT TCAACAATAA CAGAGTATAG 1620 ATAATCATTT AATCCATCAT ACATAGTAGGAAGATTCTCG TTAACAAATT GTTTAACTGC 1680 ACTGATAAAA CTGGGACTAT ATTTGATATCTTGTCTAATA AAATTAATAA CATTGTCCAA 1740 AGGATACTTT TTAACTAGAT TTATACATATCTGTTCATCA GTGAGGTTAT GCAAAACATC 1800 GTGAATAGGT GGCACATTAT ATTCATCAGATATACTAATA ACAATTTCCA GATCTATATT 1860 GTTTAATATA TTATATAGAT GGAGCACGGATCCAACGGGG ATCTCTTTAA CCACATCACG 1920 GATTTCATCA ACTGTTAAAT CTATTTTAAAGTTAATCATG TACGCGTTAA TTTTTAAAAG 1980 ATGTGTGGCT CTAACTATAT TCTCACGAATTAGCCATTCC AAGTCACTAC GTGAGAGAAG 2040 ATCGTATTCT ATCATAAGCT T 2061 (2)INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:128 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met GluLeu Val Asn Ile Phe Leu Glu Thr Asp Thr Gly Arg Val Lys 1 5 10 15 PheSer Ile Asn Asn Thr Glu Ser Val Cys Val Ser Glu Leu Ile Asn 20 25 30 LysPhe Val Glu Leu Leu Ser Glu Tyr Ile His Ile Asp Gln Ser Glu 35 40 45 PheTyr Leu Val Leu Lys Asp Lys Asp Ile Phe Tyr Phe Lys Cys Asp 50 55 60 ArgGly Ser Ile Ser Ile Val Asn Asn Glu Phe Tyr Val Phe Asp Glu 65 70 75 80Ser Leu Leu Phe Val Lys Asp Phe Thr Lys Val Thr Gly Ile Glu Phe 85 90 95Ile Val Thr Glu Ser Met Pro Thr Arg Ile Val Pro Lys Asn Asp Tyr 100 105110 Ala Val Ile Ser Val Val Thr Asn His Lys Phe Tyr Asp Gly Leu Ser 115120 125 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 688 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:3: Trp Pro Cys Ser Ser Phe Lys Asx Met Gln Lys Asp Glx Leu Asp His 15 10 15 Met Pro Ser Thr Cys Glx Val Gln Lys Lys Xaa Val Glu Pro Pro Phe20 25 30 Trp Leu Gln Tyr Asp Pro Gln Lys His Thr Phe Trp Trp Pro Glu His35 40 45 Thr Glx Leu Ala Ser Thr Gln Glu Ser His Ala His Val Ser Val Lys50 55 60 Xaa Cys Cys Thr Trp His Thr Asp Asn Ser Ala Asn Tyr His Gly Phe65 70 75 80 His Glx Glx Arg Ser Ile Val Asx Glu Xaa Asx Pro Trp His ProLeu 85 90 95 Ala Ser Glu Arg Glu Thr Pro Leu Ser His Pro Pro Leu Ser ProArg 100 105 110 Ser Gln Arg Xaa Cys Arg Ile Glu Arg Xaa Leu Cys Arg XaaLys Leu 115 120 125 Met Ile Glu Tyr Asp Leu Leu Ser Arg Ser Asp Leu GluTrp Leu Ile 130 135 140 Arg Glu Asn Ile Val Arg Ala Thr His Leu Leu LysIle Asn Ala Tyr 145 150 155 160 Met Ile Asn Phe Lys Ile Asp Leu Thr ValAsp Glu Ile Arg Asp Val 165 170 175 Val Lys Glu Ile Pro Val Gly Ser ValLeu His Leu Tyr Asn Ile Leu 180 185 190 Asn Asn Ile Asp Leu Glu Ile ValIle Ser Ile Ser Asp Glu Tyr Asn 195 200 205 Val Pro Pro Ile His Asp ValLeu His Asn Leu Thr Asp Glu Gln Ile 210 215 220 Cys Ile Asn Leu Val LysLys Tyr Pro Leu Asp Asn Val Ile Asn Phe 225 230 235 240 Ile Arg Gln AspIle Lys Tyr Ser Pro Ser Phe Ile Ser Ala Val Lys 245 250 255 Gln Phe ValAsn Glu Asn Leu Pro Thr Met Tyr Asp Gly Leu Asn Asp 260 265 270 Tyr LeuTyr Ser Val Ile Val Glu Glu Glu Leu Ile Asp Glu Tyr Lys 275 280 285 IleLys Ser Val Ala Met Phe Asn Leu Gly Tyr Glu Thr Glu Ile Asp 290 295 300Met Leu Thr Leu Asp Glu Arg Lys Phe Val Glu Thr Asn Ile Ala Tyr 305 310315 320 Tyr Asp Phe Arg Tyr Arg Arg Phe Ala Asp Glu Phe Arg Asn Phe Ile325 330 335 Met Lys Thr Glu Arg Arg Leu Ile Thr Met Pro Ser Gly Asp ArgVal 340 345 350 Arg Arg Ser Arg Arg Pro Lys Ser Leu Arg Ser Thr Ile IleLys Lys 355 360 365 Asp Thr Asp Ser Leu Glu Asp Val Leu Glu His Ile AspAsn Ala Arg 370 375 380 Arg Asn Ser Lys Leu Ser Ile Gly Asp Val Glu ArgIle Ile Ser Ser 385 390 395 400 Phe Arg Leu Asn Pro Cys Ile Val Arg ArgVal Met Leu Ser Ser Ile 405 410 415 Asp Ile Lys Thr Lys Ile Met Val IleLys Ile Val Lys Asp Trp Lys 420 425 430 Ser Cys Ser Leu Thr Leu Ser AlaIle Lys Gly Ile Met Val Thr Asp 435 440 445 Thr Ile Asn Thr Val Phe SerLys Ile Leu His Tyr His Arg Lys Ile 450 455 460 Phe Lys Tyr Ile Thr SerVal Asp Asp Lys Asp Ile Thr Ser Cys Cys 465 470 475 480 Cys Ser Arg CysIle Asn Leu Phe Tyr Arg Glu Leu Lys Ser Ile Val 485 490 495 Cys Asn LeuAsp Thr Asp Asp Gly Leu Leu Asp Arg Leu Tyr Asp Leu 500 505 510 Thr ArgTyr Ala Ile His Gly Lys Ile Asn Gln Lys Leu Ile Gly Gln 515 520 525 ArgCys Trp Gly Pro Leu Thr Glu Met Met Phe Asn Glu Val Lys Lys 530 535 540Lys Lys Leu Asn Asp Leu Met Glu His Ile Arg Ile Ser Asp Met Leu 545 550555 560 Val Tyr Gly Tyr Ser Ile Asn Glu Thr Leu Ile Pro Phe Thr Asp Ser565 570 575 Leu Ser Phe Lys Leu Ser Val Asp Thr Met Ser Val Leu Asn AspGln 580 585 590 Tyr Ala Lys Val Val Ile Phe Phe Asn Thr Ile Ile Glu TyrIle Val 595 600 605 Ala Thr Ile Tyr Tyr Arg Leu Ala Val Leu Asn Asn TyrAsp Gly Ile 610 615 620 Lys His Phe Val Ser Lys Val Leu His Thr Val IleGlu Ala Cys Gly 625 630 635 640 Val Leu Phe Ser His Ile Lys Val His AspLys Ile Glu His Glu Met 645 650 655 Glu Glu Met Val Asp Lys Gly Thr ValPro Ser Tyr Leu Tyr His Leu 660 665 670 Ser Ile Thr Thr Ile Ser Ile IleLeu Asp Asp Ile Asn Gly Thr Cys 675 680 685 (2) INFORMATION FOR SEQ IDNO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1793 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE: (A) ORGANISM: Raccoonpox virus ATCC# VR-838 (xi)SEQUENCE DESCRIPTION: SEQ ID NO:4: AAGCTTCCCG TTACTTTAGT AAAATCTTTTACAAATAACA AGGATTCATC AAATACGTAA 60 AACTCATTAT TTACTATAGA AATTGATCCCCTATCACACT TGAAATAAAA AATATCTTTA 120 TCCTTTAACA CCAAATAAAA TTCAGATTGGTCAATATGAA TGTATTCACT TAGTAATTCA 180 ACGAACTTAT TTATTAATTC TGATACACACACTGATTCTG TATTGTTTAT CGAAAACTTT 240 ACTCTTCCTG TATCAGTTTC TAAAAAAATATTAACAAGTT CCATTTATAT CATCCAATAT 300 TATTGAAATG GTGGTAATAG ACAAATGATATAGGTAAGAC GGCACAGTAC CTTTGTCGAC 360 CATCTCCTCC ATTTCATGCT CTATCTTATCATGAACTTTG ATATGTGAAA ACAATACACC 420 ACATGCTTCT ATAACAGTAT GTAACACTTTAGATACAAAA TGCTTGATAC CATCGTAATT 480 GTTCAACACG GCCAATCTAT AATAGATAGTAGCTACTATA TATTCTATGA TAGTATTGAA 540 GAAGATAACC ACCTTGGCAT ATTGGTCATTTAATACAGAC ATGGTATCAA CAGATAGCTT 600 AAACGATAGG GAATCCGTGA ATGGAATAAGCGTTTCATTG ATTGAATATC CATATACTAA 660 CATATCGGAT ATCCTGATGT GTTCCATTAAATCGTTAAGT TTTTTCTTTT TAACCTCGTT 720 GAACATCATT TCTGTTAACG GTCCCCAACATCTTTGACCA ATTAATTTTT GATTTATTTT 780 TCCATGTATG GCGTATCTAG TCAGGTCGTATAACCTATCC AATAATCCAT CATCGGTATC 840 CAAATTACAT ACTATGCTTT TCAATTCTCTATAGAAAAGA TTAATACATC TAGAACAACA 900 ACATGATGTA ATATCTTTAT CATCAACAGATGTGATATAT TTGAAGATTT TTCTATGATA 960 ATGAAGAATT TTTGAGAATA CTGTATTAATGGTGTCTGTT ACCATGATCC CTTTGATTGC 1020 TGACAGAGTC AGCGAGCATG ATTTCCAATCTTTAACAATT TTTATTACCA TTATCTTTGT 1080 TTTAATGTCT ATAGATGATA ACATAACACGTCTAACAATA CACGGATTAA GACGGAAAGA 1140 TGAAATTATT CTTTCAACAT CTCCAATGGATAACTTACTA TTTCGACGTG CATTGTCTAT 1200 ATGCTCGAGA ACATCCTCCA ACGAGTCTGTATCTTTTTTG ATTATCGTCG ATCTCAACGA 1260 TTTGGGTCGT CTAGATCGTC GGACTCTATCACCAGATGGC ATAGTTATTA GTCTTCTTTC 1320 TGTTTTCATA ATGAAATTTC TAAATTCATCTGCGAATCTT CTATATCTAA AATCATAATA 1380 TGCGATATTT GTTTCAACAA ATTTCCGTTCGTCCAATGTC AACATATCTA TCTCGGTTTC 1440 ATACCCTAAA TTGAACATGG CTACGGATTTAATTTTATAT TCATCTATCA ACTCTTCTTC 1500 AACAATAACA GAGTATAGAT AATCATTTAATCCATCATAC ATAGTAGGAA GATTCTCGTT 1560 AACAAATTGT TTAACTGCAC TGATAAAACTGGGACTATAT TTGATATCTT GTCTAATAAA 1620 ATTAATAACA TTGTCCAAAG GATACTTTTTAACTAGATTT ATACATATCT GTTCATCAGT 1680 GAGGTTATGC AAAACATCGT GAATAGGTGGCACATTATAT TCATCAGATA TACTAATAAC 1740 AATTTCCAGA TCTATATTGT TTAATATATTATATAGATGG AGCACGGAAG CTT 1793 (2) INFORMATION FOR SEQ ID NO:5: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 4491 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINALSOURCE: (A) ORGANISM: Raccoonpox virus ATCC# VR-838 (ix) FEATURE: (A)NAME/KEY: CDS (B) LOCATION: 81..1 (ix) FEATURE: (A) NAME/KEY: CDS (B)LOCATION: 855..181 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:2152..812 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 2850..2194 (ix)FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 3816..2950 (ix) FEATURE: (A)NAME/KEY: CDS (B) LOCATION: 4491..3970 (xi) SEQUENCE DESCRIPTION: SEQ IDNO:5: AAGCTTTTTA AAATGATCAC TGTAATATGA TGAATCTGTA TCATTTCTCC AAATAATGTA60 TCTGAATAGG AGATTTCTCA TATCTACTAA TGTATTTAGT TAGTAAACTA TTATTGTGTT 120AAATAAGTAT TTATGAATTA GATAACAATT TTTAAAAGAC TATGTTTGAT ACAAAAAAAA 180TTAATTTTGG AAATACTTGT CCCTCAAGTA ATCGGCAAAG CAATCTATAT AATCATATGT 240GACATTATCA TATAATACAT GAATATATAC TGGATCGTAA CTAGTTAAGA TATACACGAA 300TCCGATAACC TTTTTTACAT AATCAACAGG ATCTATTTTA AATGTATCGT ATCTAGTTCG 360CTTTATTTTT CTGAATAGAT TAATGTTGTA ATTAGGATTT CTATGTATGT CTTTTATGGT 420GTATTCCACG TAGCTACCAT AATGATCAAT AGCCAATTGT TTGTAAGTCA TTAGCTCTTT 480TATTATGTTA CATTTGGTGT TACGTAGTTC ATCGCTGTTC ATTGCCAAGA ATCTATCTAC 540AATTTTAAAT ACTTCAACGC CTGGATTATT AACCTGTTTC ATATACTGGG CTATATAATT 600ATCAATATAT CGAGATGATA CAGCACCGTC GTACATTACA TACACATCTT CTCCTGAGTT 660ATTCTGTGCT GATGGGCAGT TTAATACCTC CAGCATCTTG GTAACTTTAT TAGTATCCAT 720TTTTTTAATA TCATCGAGTA ACACGGCTGA AGAATCACCA TCCATTACGA TAGCTATAAA 780CACTAATCTA TATATTATTT TTATGTTATT TTTAAAGATC ATCAAAAACT TCATACGATA 840CTCCTTCATG GACATAATTT GTTACAAAAT GTTTATATGT AGTAAAGTAT CTGCGATTTT 900TGCAAAAATC TGGACGCATA AGACCAAAGT ATGCCAGGAA TGGAATAATA ATAGATTTAT 960CGAGTTTATC CTTTTCTACT GATTTCATTG TTAGATACAT GGTCTCGGAA GTCGGATTAT 1020GCAACATCAG TTTCGATAAT ATAACTGGAT TATTTAACCT GACACATTCG CTCATACAAG 1080TATGCCCATT AACAACTGAG TCTACGCTAA AATGATTAAA CAATAGATAG TCTATCATCG 1140TTTCGTATTC GAACAGAACA GCGTAGTACA TGGCATCTTC ACAAATTATA TCATTATCCA 1200ATAGATATTT AACACATCTT ATAGATCCTG ACTTAACAGC TGTTTTAAAA TCATTAGAAT 1260CAAATTGTCG TCCTTTATCA TTAATACTTT CTAGAATGTC ACATCTATCG TAAATGATAC 1320ACAGATTAAC TAGTTGATCT GTTATAGAAT CACTAGTTGA TATCAATTTG TTAAAGAGAG 1380ATACAGGAGT TAATGTTTTA ATGAGAATGG TAAGAGGACT ATCGCCGAAC TTGTTTTGTT 1440TATTAACATC TGTTGATGGA AGCAAAAGAT CGATTATATC TATATTCTTG ACTGCTTTAG 1500AACATGCAAT ATGGATAGGT GTATTTCCGT CATAATCTGC TCTTGAGGAA CTAATACCGA 1560GTTTCATCAT CCACTCAATT GTACAAGCTT TTGGATTCTC AGACATAAGA TGTCTATGAA 1620TATGATTTCT GCCAACTTTA TCTACTATCT TGGCCTCGAA TCCTATAGAC ATTAGTTTTT 1680TGAACACTAT TTCTGACGGA TCTGTGCATG ATAGTAATGG TCCACATCCT TCTACATCAA 1740CTGAGTTATT AATCTTAGCT CCGTGTTCCA CTAATAAATT TATTTTATCA ATTACATCAT 1800CCTCGTTTCC GGAGAGATAA TACAGTGGAG TCTTATTTTG TTTATCGCAC GCGTTTGGAT 1860TTGCACCATG CGTCAATAGC GTTGATATAA TTCTATTATT ATTGATTCTA GACGCTATAT 1920GTAATGGATA ATCACCGTCA TCATTTGCTT CATTAGGCGA GTAGCCTTTA TTGATAAGTT 1980CTTCTATGAA CTGCTCGTCT ATTCCTTTAA TTCCACAATA GATGTGTAAA ATACTATATT 2040TTCCAGATGG TATTGTTGAC TGCAATATAT TTCTAAATAC ATCTAAATTC TTATGTTTTG 2100ATTTGGCGTA AAGCGTTAGA TAATACTCGC TGTCAGGCAT GGTATCGTCC ATTGTTTCAT 2160AAAAATGATA TTTCAATTTC TACTTTCTAC TTTTTACTCT CTATAACAAA TGTCGTAACT 2220AACATTTTTT ATGTCAAGAA AGCAACTGTT TAGTTCATCT TTGAATGTTA CGCCATAACT 2280ACCTTGTGAA GCGTAATCGG AATTCCGATA AAGAAATTTA TCAACTGGAT GAACAAACTT 2340TGGATTAATA TTAATAGGAT CCATGCGGAG GTCGACACAT TTGACGCATG ATCCATTGAT 2400AGTTATAGCA CATTCTGTTT CATGATTCAT TGCTTCTGTA AGTTCTGAAT CGTATTTTGT 2460AGTTCCACAA TTCATTTCGG TACATGTTAT GGTTACACTA ATATTGTGCT GAAGTTTATC 2520TAGTCGTTGA GTAGTAAACA ACAGAACTGA TAGTTTATAA TCTTCACCTA CTCCCTCTGC 2580AGCTGCGACA AATCTCTGAT CCGTATCATA TATGGTCATA TTTATTTGTA ATCCATATCC 2640TGTCAAACCT ATGTTTGCAT CTCTACCAGT GTAATTTTCA TACATGTGAC ACTCACCCAT 2700AATAGTTTTG ATATCATAAT TAACACCAAT AGTGAGTTCG GCGGCAAAGT ACCAATAACG 2760ATAATCACTT CGAGACGGAC ATTGCGTGTC TTTGTATTCT GAATAACCAA GAGATATGAC 2820ACAAAAGAGT AAGAATAATC TGTAAACCAT CTTTAAAGAC TGCAACAGAT CTACTAGACA 2880AGAGTCAATG ATAGAAACCA CTACTAAAAA AAATAATTAT TCTATCAATT TTAACCAATT 2940GATCTTATTT TAAAATTGGG ATCTAAATAT TTTTGCAGAA TTGACATACA TGATTCTGAG 3000TTCCTTATTT TTGCTAATTA TCTCATCCAA TTTATTATTC TTGACAACAT CGAGTTCTTT 3060TATTAAATCA TCAGTCTTAT AGTCAACATG TTTTTCTATA ATCATTCTAG CTATTACAGG 3120ATCATCCAAC AATACATTTT CCAGATTAAC AGAATAGATA TTAATGTCAT ATTTGAACAG 3180AGTCCGTAGC AAATCTAAGT CTTTGTTCTC TATAGCCAAT TTGATGTCTG GGATGAAGAG 3240AAGAGAGTTA TTGGTATTTG TTGCCATCAT ATAGTCTAGT AACAGAATCA GCATATCAAC 3300ATTTCCATTT TTAATTGTGT CATGAATGCA ACTATAGAGA ATTGCCAGAT CAAAACTAGC 3360TTTTAGCTCT GAAATAAAGT ATTCAGTAAT GCTGACATCA TTAAGCATAA CCGCCTTATA 3420AAATGGGGTT TTCCATCCGG TTTTTCCATA GAACATCAGT TTCCATTTTT TCTTAATAAA 3480CAGTTTTATA GTTTGGATAT TGCCGGCCTC CACGGCGTAA TACAATGGAG TATTACCTCG 3540ATCATCAAAT TGCGAATCAT CCATCCCACT GAATAACAAA ATCTTTACTA TTTTAGTATC 3600CTCTAACTTG GCTGCCTGAT GCAATGGAAA CTCATTCTCG AGAAGATTTT TCAATACCCC 3660AGCATTAAGT AATTCACAAA CAAGACGCAC GTTATTATTA TCAATTGCAT AATACAACGC 3720CGTACGTCCA TGAATATCAG CCTTAAATAA GTCCTTTCCG CCGATGAAGC TTTTCAACTG 3780CTTAGACTTC CAGGTATTAA TTCGTGACAG ATCCATGTCT GAAACGAGAC GCTAAATACA 3840GTGTATGTTT CATTTTTTAT AATTTTGCAT TACATCAATC CTCCAGAGTC AATAATATCT 3900CTGATCGATG TGATCAATAG ATAAATGGCT ATCGCAAAAC AACACAACCA CATTTAATAA 3960AAATAATATT CAAGGAGATT CAACCTTACC AATAAATAAT ATAAATCCAG TAATATCATG 4020TCGGATGATA AACACAAATG GTTTATTGAA CTCTAGTTCT ACAGGTGCCG ATCTTCCTGT 4080AGCCAACATT ATAGTAGACG CCTCTGCTAC TGTTCCTTGT TCATCAACAT CAATTTTAGC 4140ATTCTGAAAC ATTTTATAGA TATATAATGG ATACTTGGTC ATGCGTTTAA ATGATGCATT 4200GTCTGGATTA AACATACTGG GTGCAATCAT TTCGGCTATA GTCTTAATAT CTCTCTTATT 4260TTCGATAGAA AATCTAGGAA GACTAAGATC ATACATTTTA TCCATTAATT GAGATGACCA 4320ATAATCTAAT TTTGTGGTTG TGATAGAGTC CACAAAATGC GTGATATTAT CTCCAATTGC 4380TATGTACATG CTAACATTAC TACCTTTATA TGGAAGTCTT ACCATGTCAT ATTCATAGTC 4440ATCTACAGTA ATTGTGTTTC CTTGTAATTT AGCAACTAAA TTCATTGTTG G 4491 (2)INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:26 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCEDESCRIPTION: SEQ ID NO:6: Met Val Ile Leu Gln Pro Cys Tyr Ser Met IleLeu Lys Lys Trp Ile 1 5 10 15 Leu Ile Lys Leu Pro Arg Cys Trp Arg Tyr 2025 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 224 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCEDESCRIPTION: SEQ ID NO:7: Met Ser Met Lys Glu Tyr Arg Met Lys Phe LeuMet Ile Phe Lys Asn 1 5 10 15 Asn Ile Lys Ile Ile Tyr Arg Leu Val PheIle Ala Ile Val Met Asp 20 25 30 Gly Asp Ser Ser Ala Val Leu Leu Asp AspIle Lys Lys Met Asp Thr 35 40 45 Asn Lys Val Thr Lys Met Leu Glu Val LeuAsn Cys Pro Ser Ala Gln 50 55 60 Asn Asn Ser Gly Glu Asp Val Tyr Val MetTyr Asp Gly Ala Val Ser 65 70 75 80 Ser Arg Tyr Ile Asp Asn Tyr Ile AlaGln Tyr Met Lys Gln Val Asn 85 90 95 Asn Pro Gly Val Glu Val Phe Lys IleVal Asp Arg Phe Leu Ala Met 100 105 110 Asn Ser Asp Glu Leu Arg Asn ThrLys Cys Asn Ile Ile Lys Glu Leu 115 120 125 Met Thr Tyr Lys Gln Leu AlaIle Asp His Tyr Gly Ser Tyr Val Glu 130 135 140 Tyr Thr Ile Lys Asp IleHis Arg Asn Pro Asn Tyr Asn Ile Asn Leu 145 150 155 160 Phe Arg Lys IleLys Arg Thr Arg Tyr Asp Thr Phe Lys Ile Asp Pro 165 170 175 Val Asp TyrVal Lys Lys Val Ile Gly Phe Val Tyr Ile Leu Thr Ser 180 185 190 Tyr AspPro Val Tyr Ile His Val Leu Tyr Asp Asn Val Thr Tyr Asp 195 200 205 TyrIle Asp Cys Phe Ala Asp Tyr Leu Arg Asp Lys Tyr Phe Gln Asn 210 215 220(2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 446 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCEDESCRIPTION: SEQ ID NO:8: Met Asp Asp Thr Met Pro Asp Ser Glu Tyr TyrLeu Thr Leu Tyr Ala 1 5 10 15 Lys Ser Lys His Lys Asn Leu Asp Val PheArg Asn Ile Leu Gln Ser 20 25 30 Thr Ile Pro Ser Gly Lys Tyr Ser Ile LeuHis Ile Tyr Cys Gly Ile 35 40 45 Lys Gly Ile Asp Glu Gln Phe Ile Glu GluLeu Ile Asn Lys Gly Tyr 50 55 60 Ser Pro Asn Glu Ala Asn Asp Asp Gly AspTyr Pro Leu His Ile Ala 65 70 75 80 Ser Arg Ile Asn Asn Asn Arg Ile IleSer Thr Leu Leu Thr His Gly 85 90 95 Ala Asn Pro Asn Ala Cys Asp Lys GlnAsn Lys Thr Pro Leu Tyr Tyr 100 105 110 Leu Ser Gly Asn Glu Asp Asp ValIle Asp Lys Ile Asn Leu Leu Val 115 120 125 Glu His Gly Ala Lys Ile AsnAsn Ser Val Asp Val Glu Gly Cys Gly 130 135 140 Pro Leu Leu Ser Cys ThrAsp Pro Ser Glu Ile Val Phe Lys Lys Leu 145 150 155 160 Met Ser Ile GlyPhe Glu Ala Lys Ile Val Asp Lys Val Arg Gly Asn 165 170 175 His Ile HisArg His Leu Met Ser Glu Asn Pro Lys Ala Cys Thr Ile 180 185 190 Glu TrpMet Met Lys Leu Gly Ile Ser Ser Ser Arg Ala Asp Tyr Asp 195 200 205 GlyAsn Thr Pro Ile His Ile Ala Cys Ser Lys Ala Val Lys Asn Ile 210 215 220Asp Ile Ile Asp Leu Leu Leu Pro Ser Thr Asp Val Asn Lys Gln Asn 225 230235 240 Lys Phe Gly Asp Ser Pro Leu Thr Ile Leu Ile Lys Thr Lys Thr Pro245 250 255 Val Ser Leu Phe Asn Lys Leu Ile Ser Thr Ser Asp Ser Ile ThrAsp 260 265 270 Gln Leu Val Asn Leu Cys Ile Ile Tyr Asp Arg Cys Asp IleLeu Glu 275 280 285 Ser Ile Asn Asp Lys Gly Arg Gln Phe Asp Ser Asn AspPhe Lys Thr 290 295 300 Ala Val Lys Ser Gly Ser Ile Arg Cys Val Lys TyrLeu Leu Asp Asn 305 310 315 320 Asp Ile Ile Cys Glu Asp Ala Met Tyr TyrAla Val Leu Phe Glu Tyr 325 330 335 Glu Thr Met Ile Asp Tyr Leu Leu PheAsn His Phe Ser Val Asp Ser 340 345 350 Val Val Asn Gly His Thr Cys MetSer Glu Cys Val Arg Leu Asn Asn 355 360 365 Pro Val Ile Leu Ser Lys LeuMet Leu His Asn Pro Thr Ser Glu Thr 370 375 380 Met Tyr Leu Thr Met LysSer Val Glu Lys Asp Lys Leu Asp Lys Ser 385 390 395 400 Ile Ile Ile ProPhe Leu Ala Tyr Phe Gly Leu Met Arg Pro Asp Phe 405 410 415 Cys Lys AsnArg Arg Tyr Phe Thr Thr Tyr Lys His Phe Val Thr Asn 420 425 430 Tyr ValHis Glu Gly Val Ser Tyr Glu Val Phe Asp Asp Leu 435 440 445 (2)INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:218 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCEDESCRIPTION: SEQ ID NO:9: Met Val Tyr Arg Leu Phe Leu Leu Phe Cys ValIle Ser Leu Gly Tyr 1 5 10 15 Ser Glu Tyr Lys Asp Thr Gln Cys Pro SerArg Ser Asp Tyr Arg Tyr 20 25 30 Trp Tyr Phe Ala Ala Glu Leu Thr Ile GlyVal Asn Tyr Asp Ile Lys 35 40 45 Thr Ile Met Gly Glu Cys His Met Tyr GluAsn Tyr Thr Gly Arg Asp 50 55 60 Ala Asn Ile Gly Leu Thr Gly Tyr Gly LeuGln Ile Asn Met Thr Ile 65 70 75 80 Tyr Asp Thr Asp Gln Arg Phe Val AlaAla Ala Glu Gly Val Gly Glu 85 90 95 Asp Tyr Lys Leu Ser Val Leu Leu PheThr Thr Gln Arg Leu Asp Lys 100 105 110 Leu Gln His Asn Ile Ser Val ThrIle Thr Cys Thr Glu Met Asn Cys 115 120 125 Gly Thr Thr Lys Tyr Asp SerGlu Leu Thr Glu Ala Met Asn His Glu 130 135 140 Thr Glu Cys Ala Ile ThrIle Asn Gly Ser Cys Val Lys Cys Val Asp 145 150 155 160 Leu Arg Met AspPro Ile Asn Ile Asn Pro Lys Phe Val His Pro Val 165 170 175 Asp Lys PheLeu Tyr Arg Asn Ser Asp Tyr Ala Ser Gln Gly Ser Tyr 180 185 190 Gly ValThr Phe Lys Asp Glu Leu Asn Ser Cys Phe Leu Asp Ile Lys 195 200 205 AsnVal Ser Tyr Asp Ile Cys Tyr Arg Glu 210 215 (2) INFORMATION FOR SEQ IDNO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 288 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:Met Asp Leu Ser Arg Ile Asn Thr Trp Lys Ser Lys Gln Leu Lys Ser 1 5 1015 Phe Ile Gly Gly Lys Asp Leu Phe Lys Ala Asp Ile His Gly Arg Thr 20 2530 Ala Leu Tyr Tyr Ala Ile Asp Asn Asn Asn Val Arg Leu Val Cys Glu 35 4045 Leu Leu Asn Ala Gly Val Leu Lys Asn Leu Leu Glu Asn Glu Phe Pro 50 5560 Leu His Gln Ala Ala Lys Leu Glu Asp Thr Lys Ile Val Lys Ile Leu 65 7075 80 Leu Phe Ser Gly Met Asp Asp Ser Gln Phe Asp Asp Arg Gly Asn Thr 8590 95 Pro Leu Tyr Tyr Ala Val Glu Ala Gly Asn Ile Gln Thr Ile Lys Leu100 105 110 Phe Ile Lys Lys Lys Trp Lys Leu Met Phe Tyr Gly Lys Thr GlyTrp 115 120 125 Lys Thr Pro Phe Tyr Lys Ala Val Met Leu Asn Asp Val SerIle Thr 130 135 140 Glu Tyr Phe Ile Ser Glu Leu Lys Ala Ser Phe Asp LeuAla Ile Leu 145 150 155 160 Tyr Ser Cys Ile His Asp Thr Ile Lys Asn GlyAsn Val Asp Met Leu 165 170 175 Ile Leu Leu Leu Asp Tyr Met Met Ala ThrAsn Thr Asn Asn Ser Leu 180 185 190 Leu Phe Ile Pro Asp Ile Lys Leu AlaIle Glu Asn Lys Asp Leu Asp 195 200 205 Leu Leu Arg Thr Leu Phe Lys TyrAsp Ile Asn Ile Tyr Ser Val Asn 210 215 220 Leu Glu Asn Val Leu Leu AspAsp Pro Val Ile Ala Arg Met Ile Ile 225 230 235 240 Glu Lys His Val AspTyr Lys Thr Asp Asp Leu Ile Lys Glu Leu Asp 245 250 255 Val Val Lys AsnAsn Lys Leu Asp Glu Ile Ile Ser Lys Asn Lys Glu 260 265 270 Leu Arg IleMet Tyr Val Asn Ser Ala Lys Ile Phe Arg Ser Gln Phe 275 280 285 (2)INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:173 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCEDESCRIPTION: SEQ ID NO:11: Pro Thr Met Asn Leu Val Ala Lys Leu Gln GlyAsn Thr Ile Thr Val 1 5 10 15 Asp Asp Tyr Glu Tyr Asp Met Val Arg LeuPro Tyr Lys Gly Ser Asn 20 25 30 Val Ser Met Tyr Ile Ala Ile Gly Asp AsnIle Thr His Phe Val Asp 35 40 45 Ser Ile Thr Thr Thr Lys Leu Asp Tyr TrpSer Ser Gln Leu Met Asp 50 55 60 Lys Met Tyr Asp Leu Ser Leu Pro Arg PheSer Ile Glu Asn Lys Arg 65 70 75 80 Asp Ile Lys Thr Ile Ala Glu Met IleAla Pro Ser Met Phe Asn Pro 85 90 95 Asp Asn Ala Ser Phe Lys Arg Met ThrLys Tyr Pro Leu Tyr Ile Tyr 100 105 110 Lys Met Phe Gln Asn Ala Lys IleAsp Val Asp Glu Gln Gly Thr Val 115 120 125 Ala Glu Ala Ser Thr Ile MetLeu Ala Thr Gly Arg Ser Ala Pro Val 130 135 140 Glu Leu Glu Phe Asn LysPro Phe Val Phe Ile Ile Arg His Asp Ile 145 150 155 160 Thr Gly Phe IleLeu Phe Ile Gly Lys Val Glu Ser Pro 165 170 (2) INFORMATION FOR SEQ IDNO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 672 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE: (A) ORGANISM: Raccoonpox virus ATCC# VR-838 (ix)FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 95..547 (xi) SEQUENCEDESCRIPTION: SEQ ID NO:12: TATAACAGCT CGTGGAGTTG CTATATTATT TTTATGTTTTATTGATAATT GTAAAAAAAA 60 CACACAATTA AATCGATAAG ATTGGGTAAT AACCATGGAGAGTAAATTAG ATTACGAGGA 120 CGCTGTTTTT TACTTTGTAT ACGACGACGA GTTATGCAGTCGTGACCTCA TTATCGATCT 180 AATTGATGAA TACGTCACGT GGAGAAATTA TATTATTGTATTCGGTAAAG ATATTAACAA 240 ATGCGGAAGA CTATACAAGG AGTTGATGAA ATTTGACGATGTTGCCATTA ATTACTATGG 300 GATGGATAAG ATCAATGAGA TTATTGATAC TATGAATGTCGGAGATAGAT ATATTAATCT 360 TAAAGAAGTC CATGATCAGG AAAGTCTGTT TGCCACCATCGGTATATGTG CCAAAATCAC 420 TGAAAATTGG GGTTATAAAA ATTTTTCAGA ATCTAGATTTCAATCATTAG GAAATATTAA 480 AGATTTGATG ACTGATGACA ACATAAATAC GTTGATGATTTTTCTAGAAA AAAAATTAGC 540 AGTATAATAT AGTTTTTTAT AAAAAGCATT AAATAGACTCGTATTATATT TTTGTTAAAA 600 AATTCATTAA CCCATTATAT ATTTTAAAGT CTTATATGTCACAAACATGA AAACTGTAAT 660 TCCTATAAGC TT 672 (2) INFORMATION FOR SEQ IDNO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 150 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:Met Glu Ser Lys Leu Asp Tyr Glu Asp Ala Val Phe Tyr Phe Val Tyr 1 5 1015 Asp Asp Glu Leu Cys Ser Arg Asp Leu Ile Ile Asp Leu Ile Asp Glu 20 2530 Tyr Val Thr Trp Arg Asn Tyr Ile Ile Val Phe Gly Lys Asp Ile Asn 35 4045 Lys Cys Gly Arg Leu Tyr Lys Glu Leu Met Lys Phe Asp Asp Val Ala 50 5560 Ile Asn Tyr Tyr Gly Met Asp Lys Ile Asn Glu Ile Ile Asp Thr Met 65 7075 80 Asn Val Gly Asp Arg Tyr Ile Asn Leu Lys Glu Val His Asp Gln Glu 8590 95 Ser Leu Phe Ala Thr Ile Gly Ile Cys Ala Lys Ile Thr Glu Asn Trp100 105 110 Gly Tyr Lys Asn Phe Ser Glu Ser Arg Phe Gln Ser Leu Gly AsnIle 115 120 125 Lys Asp Leu Met Thr Asp Asp Asn Ile Asn Thr Leu Met IlePhe Leu 130 135 140 Glu Lys Lys Leu Ala Val 145 150 (2) INFORMATION FORSEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Raccoonpox virusATCC# VR-838 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CCGAAGCTTCCGTGCTCCAT CTATATAATA TATTAAAC 38 (2) INFORMATION FOR SEQ ID NO:15: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINALSOURCE: (A) ORGANISM: Raccoonpox virus ATCC# VR-838 (xi) SEQUENCEDESCRIPTION: SEQ ID NO:15: AATAAGCTTC CCGTTACTTT AGTAAAATCT TTTACAAA 38(2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM:Swine influenza virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:CCGAGGATCC GGCAATACTA TTAGTCTTGC TATGTACATT 40 (2) INFORMATION FOR SEQID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE: (A) ORGANISM: Swine influenza virus (xi) SEQUENCEDESCRIPTION: SEQ ID NO:17: CTCTGGGATC CTAATTTTAA ATACATATTC TGCACTGTA 39(2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM:Swine influenza virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:CCGAGGATCC GGCAATACTA TTAGTCTTGC TATGTACAT 39 (2) INFORMATION FOR SEQ IDNO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)ORIGINAL SOURCE: (A) ORGANISM: Swine influenza virus (xi) SEQUENCEDESCRIPTION: SEQ ID NO:19: CTCTGGATCC TAATTTAAAT ACATATTCTG CACTGTA 37(2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM:Swine influenza virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:AATGAATTCA AATCAAAAAA TAATAACCAT TGGGTCAAT 39 (2) INFORMATION FOR SEQ IDNO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)ORIGINAL SOURCE: (A) ORGANISM: Swine influenza virus (xi) SEQUENCEDESCRIPTION: SEQ ID NO:21: GGAAGATCTA CTTGTCAATG GTGAATGGCA GATCAG 36(2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM:Porcine respiratory reproductive syndrome virus (xi) SEQUENCEDESCRIPTION: SEQ ID NO:22: TTCGAATTCG GCTAATAGCT GTACATTCCT CCATATTT 38(2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM:Porcine respiratory reproductive syndrome virus (xi) SEQUENCEDESCRIPTION: SEQ ID NO:23: GGGGATCCTA TCGCCGTACG GCACTGAGGG 30 (2)INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Porcinerespiratory reproductive syndrome virus (xi) SEQUENCE DESCRIPTION: SEQID NO:24: CCGAATTCGG CTGCGTCCCT TCTTTTCCTC ATGG 34 (2) INFORMATION FORSEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Porcine respiratoryreproductive syndrome virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:CTGGATCCTT CAAATTGCCA ACAGAATGGC AAAAAGAC 38 (2) INFORMATION FOR SEQ IDNO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)ORIGINAL SOURCE: (A) ORGANISM: Porcine respiratory reproductive syndromevirus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: TTGAATTCGT TGGAGAAATGCTTGACCGCG GGC 33 (2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINALSOURCE: (A) ORGANISM: Porcine respiratory reproductive syndrome virus(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GAAGGATCCT AAGGACGACCCCATTGTTCC GCTG 34 (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 201 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINALSOURCE: (A) ORGANISM: Escherichia coli (xi) SEQUENCE DESCRIPTION: SEQ IDNO:28: AGATCTTCCA TAATTAATTA ACCCTCGACT CTAGATTTTT TTTTTTTTTT TTTTTGGCAT60 ATAAATAGAT CTGTATCCTA AAATTGAATT GTAATTATCG ATAATAAATG AATTCGGATC 120CATAATTAAT TAATTTTTAT CCCGGCGCGC CGGGTCGAGG GTCGACCTGC AGGGCGGCCG 180CGGCCCTCGA GGCCAAGCTT G 201 (2) INFORMATION FOR SEQ ID NO:29: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 189 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINALSOURCE: (A) ORGANISM: Escherichia coli (xi) SEQUENCE DESCRIPTION: SEQ IDNO:29: AGATCTTCCA TAATTAATTA ACCCTCGACT CTAGATTTTT TTTTTTTTTT TTTTTGGCAT60 ATAAATAGAT CTGTATCCTA AAATTGAATT GTAATTATCG ATAATAAATG AATTCGGATC 120CATAATTAAT TAATTTTTAT CCCGGCGCGC CGGGTCGAGG GTCGACCTGC AGGGCGGCCG 180CGGCCCTCG 189 (2) INFORMATION FOR SEQ ID NO:30: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 90 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINALSOURCE: (A) ORGANISM: Raccoonpox virus ATCC# VR-838 (xi) SEQUENCEDESCRIPTION: SEQ ID NO:30: ATAGGTAAGA CGGCACAGTA CCTTTGTCGA CCATCTCCTCCACATTTATA TCATCCAATA 60 TTATTGAAAT GGTGGTAATA GACAAATGAT 90 (2)INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Porcineparvovirus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: CTAAGGATCCGAGTGAAAAT GTGGAACAAC ACAACCC 37 (2) INFORMATION FOR SEQ ID NO:32: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINALSOURCE: (A) ORGANISM: Porcine parvovirus (xi) SEQUENCE DESCRIPTION: SEQID NO:32: GTGGATCCTA GTATAATTTT CTTGGTATAA GTTGTGAA 38 (2) INFORMATIONFOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2375 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..738 (ix)FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 799..2375 (ix) FEATURE: (A)NAME/KEY: unsure (B) LOCATION: 1106 (ix) FEATURE: (A) NAME/KEY: unsure(B) LOCATION: 1109 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: AAGCTTGTTAAACATGGCGA TTTATGGATA GCCAATTATA CATCTAAAGA CAGTCACCGT 60 AGGTATTTGTGTACTGTAAC GACAAAGAAT GGAGATTGTG TTCAGGGTAT AGTTAGATCT 120 CATATTAGGAAACCTTCATG TATTCCACAA ACCTATGAAC TAGGTACTCA CGATAAGTAT 180 GGTATAGACTTATATTGTGG AATTCTTTAC GCAAACCATT ATGATAATAT AACTTGGTAT 240 AAAAATAATCAAGAAATCAA TATCGACGGT ATTAAGTATT CTCAATCGGA TAAGAATTTA 300 ATTATTCACAGTCCAGAGTT AGAAGATAGT GGAAGATACA ATTGTTATGT TCACTACAAT 360 GACGTTAAAAACAAGGACGA TATCATCGTA TCAAGATGTA GAATGCTTAC AGTTATACCG 420 TCACAAGACCACAGGTTTAA ACTAATACTA GATCCGAAAA TCAATGCGAC AATAGGAAAA 480 CCTGCCAACATAACATGTAT GGCGGTTACT AGTTCGCTAT CGATTGATGA TTTACTAATA 540 GAATGGGAAGATCCATCCGG ATGGTTTATA GGATTCGATT TTGATGTATA TGCGGTTTTA 600 TATAGTGAAGGTGGTGGTAT CACCAGGTCG ACATTGTATT TTGAAAATGT TACTGAAGAA 660 TATATAGGTAATACATATAC CTGTCGTGGA CACAACTATT ATTTTCAGAA AACCCTTACG 720 ACCACAGTAGTATTGGAGTA AACAACTAAC ATTTTTATAT TACTGAAATT TAACGTATAA 780 TCCTCGTTTCTATATAAAAT GGATGAAGAT AGGATGCTAC TAACTAAGTA TATGTATCTC 840 ACTGATAGAGAACATATAAA TATAGACTCT GTTAAACGGT TATGTGAAAT ATCGGATCCT 900 AATGCGTGTTATAGATCAGG ATGTACCGCT TTACACGAGT ACTTTTATAA TTGTAGATCA 960 ATCAAAGGAAAATACGATTA TAGGTATAAT GGTTATTATA AATATTATTA TTCAGGAGAT 1020 TATGAAAACTATAACAGTAA TTATGATCAT GCTATAGATA TGACAGACGA TGACGACACG 1080 GAAGATGATGAATCATCTGA AACAANATNT GAATTTTATG ATGAAACACA AGATCAAAAT 1140 AATCAGCTAATATGTTCTAA TATTAAACTC ACAAATAGAG TAGATGATTT CGTAGATGAA 1200 TTCTATGGTTACGATCAGAA TATCGACATA AATGATTATG TAGATAAATC TATAAAACAT 1260 GTAGTATATGGAAGCGATAA TAGGCGAATG CGATGGTGTG ATGTATGGAG ATGTCACCAT 1320 AGCGGATCATACAGATTCGG TAAGGAATGT ATAGACAATA TATATGAAGA TAATAATACA 1380 AAAATAGAATTCGATAATTT CGATTCGTTG TCAAACCTTA CTGATGCGGA ATATGTAATT 1440 CGGGTAACTAGAGATGCGTC TACACAAATA TGGGAAAAGA AATCAGTGTT AGATAGATAC 1500 ATGGAGTCATATAGCCGTAA CCGATATAGT AAACATTCTG TCTTTAAGGG ATTTTCTGAT 1560 TACGTTAGAAAAAATGATTT AGATATGAAT ATTGTTAAAG AACTACTTTC GAACGGCGCA 1620 TCGCTGACAATCAATGATGG AAGTCGTTGG GACCCCATCT TAGTGTACTT CAGAAGAACT 1680 ATAATGAATTTAGACATGAT CGATATTATT AACAATCATA CAACTATAGA TGAACGAAAG 1740 TATATAGTACACATCTATCT AAACAATTAC AGAAATTTCG ATTACCCATT CTTTAGGAAG 1800 TTAGTCATAACCAATAAACA TTGTCTTAAG AATTATTATA CTAATCATGA TGATATATAC 1860 GGCACACCACTTCATATGTT ATCATCTAAT AAAAAATTAA TAACGCCTAA TTACATTAAA 1920 TTATTGGTATATAACGGAAA TGATATAAAC GCGCGAGGGA ATGAGTCATA TATGAGAACT 1980 CCATTACATCATTATTTAAG TAAAATGGTT TATCACGATA GAGAATATGA TGTTGGATAT 2040 TATAATGAAAAGATTATAGA TGCCTTTATA GAATTAGGAG CCGATCTAAC GATTGCGGAT 2100 AATAATGAGATGATACCAGT AATTCATGCT ATTCATAGAA ATTCCGCATG TGATGGTTAC 2160 ATCAATACCAATAATATAAA GATAATAAGT AAACTACTTA ATCTCAGTAG ACACGCACCG 2220 CATAATCTATTTAGGGATCG CGTAATGCAT GATTATATAA TTAATTCATA TACTAATCTT 2280 GAATGTTTGGATATTATTAG ATCACTGGAT GGATTTGATA TTAATTGTTA CTTTAACGGA 2340 CATACACCACTTCATTGTGC CATAATTAAA AGCTT 2375 (2) INFORMATION FOR SEQ ID NO:34: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 246 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: Lys Leu Val LysHis Gly Asp Leu Trp Ile Ala Asn Tyr Thr Ser Lys 1 5 10 15 Asp Ser HisArg Arg Tyr Leu Cys Thr Val Thr Thr Lys Asn Gly Asp 20 25 30 Cys Val GlnGly Ile Val Arg Ser His Ile Arg Lys Pro Ser Cys Ile 35 40 45 Pro Gln ThrTyr Glu Leu Gly Thr His Asp Lys Tyr Gly Ile Asp Leu 50 55 60 Tyr Cys GlyIle Leu Tyr Ala Asn His Tyr Asp Asn Ile Thr Trp Tyr 65 70 75 80 Lys AsnAsn Gln Glu Ile Asn Ile Asp Gly Ile Lys Tyr Ser Gln Ser 85 90 95 Asp LysAsn Leu Ile Ile His Ser Pro Glu Leu Glu Asp Ser Gly Arg 100 105 110 TyrAsn Cys Tyr Val His Tyr Asn Asp Val Lys Asn Lys Asp Asp Ile 115 120 125Ile Val Ser Arg Cys Arg Met Leu Thr Val Ile Pro Ser Gln Asp His 130 135140 Arg Phe Lys Leu Ile Leu Asp Pro Lys Ile Asn Ala Thr Ile Gly Lys 145150 155 160 Pro Ala Asn Ile Thr Cys Met Ala Val Thr Ser Ser Leu Ser IleAsp 165 170 175 Asp Leu Leu Ile Glu Trp Glu Asp Pro Ser Gly Trp Phe IleGly Phe 180 185 190 Asp Phe Asp Val Tyr Ala Val Leu Tyr Ser Glu Gly GlyGly Ile Thr 195 200 205 Arg Ser Thr Leu Tyr Phe Glu Asn Val Thr Glu GluTyr Ile Gly Asn 210 215 220 Thr Tyr Thr Cys Arg Gly His Asn Tyr Tyr PheGln Lys Thr Leu Thr 225 230 235 240 Thr Thr Val Val Leu Glu 245 (2)INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:525 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCEDESCRIPTION: SEQ ID NO:35: Met Asp Glu Asp Arg Met Leu Leu Thr Lys TyrMet Tyr Leu Thr Asp 1 5 10 15 Arg Glu His Ile Asn Ile Asp Ser Val LysArg Leu Cys Glu Ile Ser 20 25 30 Asp Pro Asn Ala Cys Tyr Arg Ser Gly CysThr Ala Leu His Glu Tyr 35 40 45 Phe Tyr Asn Cys Arg Ser Ile Lys Gly LysTyr Asp Tyr Arg Tyr Asn 50 55 60 Gly Tyr Tyr Lys Tyr Tyr Tyr Ser Gly AspTyr Glu Asn Tyr Asn Ser 65 70 75 80 Asn Tyr Asp His Ala Ile Asp Met ThrAsp Asp Asp Asp Thr Glu Asp 85 90 95 Asp Glu Ser Ser Glu Thr Xaa Xaa GluPhe Tyr Asp Glu Thr Gln Asp 100 105 110 Gln Asn Asn Gln Leu Ile Cys SerAsn Ile Lys Leu Thr Asn Arg Val 115 120 125 Asp Asp Phe Val Asp Glu PheTyr Gly Tyr Asp Gln Asn Ile Asp Ile 130 135 140 Asn Asp Tyr Val Asp LysSer Ile Lys His Val Val Tyr Gly Ser Asp 145 150 155 160 Asn Arg Arg MetArg Trp Cys Asp Val Trp Arg Cys His His Ser Gly 165 170 175 Ser Tyr ArgPhe Gly Lys Glu Cys Ile Asp Asn Ile Tyr Glu Asp Asn 180 185 190 Asn ThrLys Ile Glu Phe Asp Asn Phe Asp Ser Leu Ser Asn Leu Thr 195 200 205 AspAla Glu Tyr Val Ile Arg Val Thr Arg Asp Ala Ser Thr Gln Ile 210 215 220Trp Glu Lys Lys Ser Val Leu Asp Arg Tyr Met Glu Ser Tyr Ser Arg 225 230235 240 Asn Arg Tyr Ser Lys His Ser Val Phe Lys Gly Phe Ser Asp Tyr Val245 250 255 Arg Lys Asn Asp Leu Asp Met Asn Ile Val Lys Glu Leu Leu SerAsn 260 265 270 Gly Ala Ser Leu Thr Ile Asn Asp Gly Ser Arg Trp Asp ProIle Leu 275 280 285 Val Tyr Phe Arg Arg Thr Ile Met Asn Leu Asp Met IleAsp Ile Ile 290 295 300 Asn Asn His Thr Thr Ile Asp Glu Arg Lys Tyr IleVal His Ile Tyr 305 310 315 320 Leu Asn Asn Tyr Arg Asn Phe Asp Tyr ProPhe Phe Arg Lys Leu Val 325 330 335 Ile Thr Asn Lys His Cys Leu Lys AsnTyr Tyr Thr Asn His Asp Asp 340 345 350 Ile Tyr Gly Thr Pro Leu His MetLeu Ser Ser Asn Lys Lys Leu Ile 355 360 365 Thr Pro Asn Tyr Ile Lys LeuLeu Val Tyr Asn Gly Asn Asp Ile Asn 370 375 380 Ala Arg Gly Asn Glu SerTyr Met Arg Thr Pro Leu His His Tyr Leu 385 390 395 400 Ser Lys Met ValTyr His Asp Arg Glu Tyr Asp Val Gly Tyr Tyr Asn 405 410 415 Glu Lys IleIle Asp Ala Phe Ile Glu Leu Gly Ala Asp Leu Thr Ile 420 425 430 Ala AspAsn Asn Glu Met Ile Pro Val Ile His Ala Ile His Arg Asn 435 440 445 SerAla Cys Asp Gly Tyr Ile Asn Thr Asn Asn Ile Lys Ile Ile Ser 450 455 460Lys Leu Leu Asn Leu Ser Arg His Ala Pro His Asn Leu Phe Arg Asp 465 470475 480 Arg Val Met His Asp Tyr Ile Ile Asn Ser Tyr Thr Asn Leu Glu Cys485 490 495 Leu Asp Ile Ile Arg Ser Leu Asp Gly Phe Asp Ile Asn Cys TyrPhe 500 505 510 Asn Gly His Thr Pro Leu His Cys Ala Ile Ile Lys Ser 515520 525 (2) INFORMATION FOR SEQ ID NO:36: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 3295 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM:Porcine reproductive respiratory virus (Eichelberger strain) (ix)FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..738 (ix) FEATURE: (A)NAME/KEY: CDS (B) LOCATION: 799..2375 (ix) FEATURE: (A) NAME/KEY: CDS(B) LOCATION: 1..738 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:799..2375 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..738 (ix)FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 799..2375 (xi) SEQUENCEDESCRIPTION: SEQ ID NO:36: GAATTCCCGG GCCCTGTCAT TGAACCAACT TTGGGCCTGAACTGAAATGA AATGGGGGCT 60 ATGCAAAGCC TTTTCGACAA AATTGGCCAA CTTTTTGTGGATGCTTTCAC GGAATTTTTG 120 GTGTCCATTG TTGATATCAT CATATTTTTG GCCATTTTGTTTGGCTTCAC CATCGCAGGT 180 TGGCTGGTGG TCTTCTGCAT CCGATTGGTT TGCTCCGCGGTACTCCGTGC GCGCCCTACC 240 ATTCACCCTG AGCAATTACA GAAGATCCTA TGAAGCCTTTCTTTCTCAGT GCCAGGTGGA 300 CATTCCCACC TGGGGAATCA AACATCCCTT GGGGATGTTTTGGCACCATA AGGTGTCAAC 360 CCTGATTGAT GAAATGGTGT CGCGTCGAAT GTACCGCATCATGGAAAAAG CAGGACAGGC 420 TGCCTGGAAA CAGGTGGTGA GGGAGGTTAC GCTGTCTCGCATTAGTGGTT TGGACGTGGT 480 GGCTCATTTT CAGCATCTTG CCGCCATTGA AGCCGAGACCTGTAAATATT TGGCCTCTCG 540 GCTGCCCATG CTACACAACC TGCGCATGAC AGGGTCAAATGTAACCATAG TGTATAATAG 600 CACTTCAAAT CAGGTGTTTG CTATTTTTCC AACCCCTGGGTCCCGGCCAA AGCTTCATGA 660 TTTTCAGCAA TGGCTAATAG CTGTGCACTC CTCCATATTTTCCTCCGTAG CGGCTTCTTG 720 TACTCTTTTT GTTGTGCTGT GGTTGCGGAT CCCAATGCTACGTTCTGTTT TTGGTTTCCA 780 CTGGTTAGGG GCAATTTTTC TTTCGAACTC ACGGTGAATTACACAGTGTG CCCACCTTGC 840 CTCACCCGGC AAGCAGCCGC TGAGATCTAC GAACCCGGCAGGTCTCTTTG GTGCAGGATA 900 GGGCATGACC GATGTGGTGA GGACGATCAT GACGAACTAGGGTTCATGGT TCCGCCTGGC 960 CTCTCCAGCG AAGGCCACTT GACCAGTGTT TACGCTTGGTTGGCGTTCCT GTCCTTCAGC 1020 TACACGGCCC AGTTCCATCC CGAGATATTT GGGATAGGGAATGTGAGTAA AGTTTATGTT 1080 GACATCAAGC ACCAATTCAT CTGCGCCGAA CACGACGGGCGGAACGCCAC CCTGCTTCGC 1140 CATGACAATA TTTCAGCCGT GTTTCAGACC TACTACCAACATCAGGTCGA TGGCGGCAAT 1200 TGGTTTCACC TGGAATGGCT GCGTCCCTTC TTTTCCTCTTGGTTGGTTTT AAATGTTTCG 1260 TGGTTTCTCA GGCGTTCGCC TGCAAGCCAT GTTTCAGTTCGAGTCTTTCA GACATCAAAA 1320 CCAACACTAC CGCAGCATCA AACTTTGTTG TCCTCCAGGACGTCAGCTGC CTTAGGCATG 1380 GCGACCCGTC CTCTCCGGCG ATTCGCAAAA GCCCTCAGTGCCGTACGGCG ATAGGAACAC 1440 CCGTGTATAT CACCACCACA GCCAATGTGA CAGATGAGAATTATTTACAT TCTTCTGATC 1500 TCCTCATGCT TTCTTCTTGC CTTTTCTATG CTTCTGAAATGAGTGAAAAG GGGTTCAAGG 1560 TGGTATTTGG CAATGTGTCA GGCATCGTGG CTGTGTGTGTCAACTTTACC AGCTACGTCC 1620 AACATGTCAA GGAGTTCACC CAACGCTCCT TGGTGGTCGATCATGTGCGG CTGCTTCATT 1680 TCATGACACC TGAGACCATG AGGTGGGCAA CCGTTTTAGCCTGTCTTTTT GCCATCCTGC 1740 TGGCAATTTG AATGTTCAAG TATGTTGGGG AAATGCTTGACCGCGGGCTG TTGCTCGCGA 1800 TTGCTTTCTT TGTGGTTTAT CGTGCCGTTC TGGTTTGCTGTGCTCGTCAA CGCCAACAGC 1860 AGCAGCAGCT CCCATTTTCA GTCGATTTAT AACTTGACGCTATGTGAGCT GAATGGCACA 1920 AATTGGCTGG CTGAAAAATT TGATTGGGCA GTGGAGACTTTTGTCATCTT TCCCGTGTTG 1980 ACTCACATTG TTTCCTATGG TGCACTTACC ACCAGCCATTTCCTTGACAC AGTTGGTCTG 2040 GTTGCTGTGT CCACCGCCGG TTTTTTTCAC GGGCGGTATGTCTTGAGTAG CATCTACGCG 2100 GTCTGTGCCC TGGCTGCGTT GATTTGCTTC GTCATCAGATTTGCGAAGAA CTGCATGTCC 2160 TGGCGCTACT CATGTACCAG ATATACCAAC TTCCTTCTAGATACTAAGGG CAGACTCTAT 2220 CGTTGGCGGT CGCCTGTTAT CATAGAGAAA GGGGGTAAGGTTGAGGTCGG AGGCCACCTG 2280 ATCGACCTCA AAAGAGTTGT GCTTGATGGT TCCGTGGCGACTCCTTTAAC CAGAGTTTCA 2340 GCTGAACAAT GGGGTCGTCC CTAGACGACT TTTGCAATGATAGCACGGCT CCGCAAAAAG 2400 TGCTTCTGGC GTTTTCCATC ACCTACACGC CAGTGATGATATATGCTCTA AAGGTAAGTC 2460 GCGGCCGACT GCTAGGGCTT CTGCACCTTT TGATCTTTCTGAATTGTGCT TTTACCTTCG 2520 GGTACATGAC ATTCGTGCAC TTTCAGAGCA CAAATAAGGTCGCGCTCACT ATGGGAGCAG 2580 TAGTTGCACT TCTTTGGGGG GTGTACTCAG CCATAGAAACCTGGAAATTC ATCACCTCCA 2640 GATGCCGTTT GTGCTTGCTA GGCCGCAAGT ACATCCTGGCCCCTGCCCAC CACGTCGAAA 2700 GTGCCGCGGG CTTTCATCCG ATTGCGGCAA ATGATAACCACGCATTTGTC GTCCGGCGTC 2760 CCGGCTCCAC TACGGTTAAC GGCACATTGG TGCCCGGGTTGAAAAGCCTC GTGTTGGGTG 2820 GCAGAAAAGC TGTTAAACAG GGAGTGGTAA ACCTTGTCAAATATGCCAAA TAACAACGGC 2880 AAGCAGCAAA AGAAAAAGAA GGGGAATGGC CAGCCAGTCAATCAGCTGTG CCAAATGCTG 2940 GGTAAGATCA TCGCCCAGCA AAACCAGTCC AGAGGCAAGGGACCGGGGAA GAAAAATAAG 3000 AAGAAAAACC CGGAGAAGCC CCATTTTCCT CTAGCGACCGAAGATGACGT CAGGCATCAC 3060 TTCACCCCTA GTGAGCGGCA ATTGTGTCTG TCGTCGATCCAGACTGCCTT TAACCAGGGC 3120 GCTGGAACTT GTACCCTGTC AGATTCAGGG AGGATAAGTTACACTGTGGA GTTTAGTTTG 3180 CCGACGCATC ATACTGTGCG TCTGATTCGC GCTACAGCATCACCCACAGC GTGATGGGCT 3240 GACATTCTTG AAGCACCTCA GTGTTTGAAT TGGAAGAATGCGTGGTGAAG GATCC 3295 (2) INFORMATION FOR SEQ ID NO:37: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 256 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: Met Lys Trp Gly LeuCys Lys Ala Phe Ser Thr Lys Leu Ala Asn Phe 1 5 10 15 Leu Trp Met LeuSer Arg Asn Phe Trp Cys Pro Leu Leu Ile Ser Ser 20 25 30 Tyr Phe Trp ProPhe Cys Leu Ala Ser Pro Ser Gln Val Gly Trp Trp 35 40 45 Ser Ser Ala SerAsp Trp Phe Ala Pro Arg Tyr Ser Val Arg Ala Leu 50 55 60 Pro Phe Thr LeuSer Asn Tyr Arg Arg Ser Tyr Glu Ala Phe Leu Ser 65 70 75 80 Gln Cys GlnVal Asp Ile Pro Thr Trp Gly Ile Lys His Pro Leu Gly 85 90 95 Met Phe TrpHis His Lys Val Ser Thr Leu Ile Asp Glu Met Val Ser 100 105 110 Arg ArgMet Tyr Arg Ile Met Glu Lys Ala Gly Gln Ala Ala Trp Lys 115 120 125 GlnVal Val Arg Glu Val Thr Leu Ser Arg Ile Ser Gly Leu Asp Val 130 135 140Val Ala His Phe Gln His Leu Ala Ala Ile Glu Ala Glu Thr Cys Lys 145 150155 160 Tyr Leu Ala Ser Arg Leu Pro Met Leu His Asn Leu Arg Met Thr Gly165 170 175 Ser Asn Val Thr Ile Val Tyr Asn Ser Thr Ser Asn Gln Val PheAla 180 185 190 Ile Phe Pro Thr Pro Gly Ser Arg Pro Lys Leu His Asp PheGln Gln 195 200 205 Trp Leu Ile Ala Val His Ser Ser Ile Phe Ser Ser ValAla Ala Ser 210 215 220 Cys Thr Leu Phe Val Val Leu Trp Leu Arg Ile ProMet Leu Arg Ser 225 230 235 240 Val Phe Gly Phe His Trp Leu Gly Ala IlePhe Leu Ser Asn Ser Arg 245 250 255 (2) INFORMATION FOR SEQ ID NO:38:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 254 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Met Ala AsnSer Cys Ala Leu Leu His Ile Phe Leu Arg Ser Gly Phe 1 5 10 15 Leu TyrSer Phe Cys Cys Ala Val Val Ala Asp Pro Asn Ala Thr Phe 20 25 30 Cys PheTrp Phe Pro Leu Val Arg Gly Asn Phe Ser Phe Glu Leu Thr 35 40 45 Val AsnTyr Thr Val Cys Pro Pro Cys Leu Thr Arg Gln Ala Ala Ala 50 55 60 Glu IleTyr Glu Pro Gly Arg Ser Leu Trp Cys Arg Ile Gly His Asp 65 70 75 80 ArgCys Gly Glu Asp Asp His Asp Glu Leu Gly Phe Met Val Pro Pro 85 90 95 GlyLeu Ser Ser Glu Gly His Leu Thr Ser Val Tyr Ala Trp Leu Ala 100 105 110Phe Leu Ser Phe Ser Tyr Thr Ala Gln Phe His Pro Glu Ile Phe Gly 115 120125 Ile Gly Asn Val Ser Lys Val Tyr Val Asp Ile Lys His Gln Phe Ile 130135 140 Cys Ala Glu His Asp Gly Arg Asn Ala Thr Leu Leu Arg His Asp Asn145 150 155 160 Ile Ser Ala Val Phe Gln Thr Tyr Tyr Gln His Gln Val AspGly Gly 165 170 175 Asn Trp Phe His Leu Glu Trp Leu Arg Pro Phe Phe SerSer Trp Leu 180 185 190 Val Leu Asn Val Ser Trp Phe Leu Arg Arg Ser ProAla Ser His Val 195 200 205 Ser Val Arg Val Phe Gln Thr Ser Lys Pro ThrLeu Pro Gln His Gln 210 215 220 Thr Leu Leu Ser Ser Arg Thr Ser Ala AlaLeu Gly Met Ala Thr Arg 225 230 235 240 Pro Leu Arg Arg Phe Ala Lys AlaLeu Ser Ala Val Arg Arg 245 250 (2) INFORMATION FOR SEQ ID NO:39: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 178 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Met Ala Ala SerLeu Leu Phe Leu Leu Val Gly Phe Lys Cys Phe Val 1 5 10 15 Val Ser GlnAla Phe Ala Cys Lys Pro Cys Phe Ser Ser Ser Leu Ser 20 25 30 Asp Ile LysThr Asn Thr Thr Ala Ala Ser Asn Phe Val Val Leu Gln 35 40 45 Asp Val SerCys Leu Arg His Gly Asp Pro Ser Ser Pro Ala Ile Arg 50 55 60 Lys Ser ProGln Cys Arg Thr Ala Ile Gly Thr Pro Val Tyr Ile Thr 65 70 75 80 Thr ThrAla Asn Val Thr Asp Glu Asn Tyr Leu His Ser Ser Asp Leu 85 90 95 Leu MetLeu Ser Ser Cys Leu Phe Tyr Ala Ser Glu Met Ser Glu Lys 100 105 110 GlyPhe Lys Val Val Phe Gly Asn Val Ser Gly Ile Val Ala Val Cys 115 120 125Val Asn Phe Thr Ser Tyr Val Gln His Val Lys Glu Phe Thr Gln Arg 130 135140 Ser Leu Val Val Asp His Val Arg Leu Leu His Phe Met Thr Pro Glu 145150 155 160 Thr Met Arg Trp Ala Thr Val Leu Ala Cys Leu Phe Ala Ile LeuLeu 165 170 175 Ala Ile (2) INFORMATION FOR SEQ ID NO:40: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 200 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: Met Leu Gly Lys CysLeu Thr Ala Gly Cys Cys Ser Arg Leu Leu Ser 1 5 10 15 Leu Trp Phe IleVal Pro Phe Trp Phe Ala Val Leu Val Asn Ala Asn 20 25 30 Ser Ser Ser SerSer His Phe Gln Ser Ile Tyr Asn Leu Thr Leu Cys 35 40 45 Glu Leu Asn GlyThr Asn Trp Leu Ala Glu Lys Phe Asp Trp Ala Val 50 55 60 Glu Thr Phe ValIle Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly 65 70 75 80 Ala Leu ThrThr Ser His Phe Leu Asp Thr Val Gly Leu Val Ala Val 85 90 95 Ser Thr AlaGly Phe Phe His Gly Arg Tyr Val Leu Ser Ser Ile Tyr 100 105 110 Ala ValCys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Phe Ala 115 120 125 LysAsn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe 130 135 140Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile 145 150155 160 Ile Glu Lys Gly Gly Lys Val Glu Val Gly Gly His Leu Ile Asp Leu165 170 175 Lys Arg Val Val Leu Asp Gly Ser Val Ala Thr Pro Leu Thr ArgVal 180 185 190 Ser Ala Glu Gln Trp Gly Arg Pro 195 200 (2) INFORMATIONFOR SEQ ID NO:41: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 174 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ IDNO:41: Met Gly Ser Ser Leu Asp Asp Phe Cys Asn Asp Ser Thr Ala Pro Gln 15 10 15 Lys Val Leu Leu Ala Phe Ser Ile Thr Tyr Thr Pro Val Met Ile Tyr20 25 30 Ala Leu Lys Val Ser Arg Gly Arg Leu Leu Gly Leu Leu His Leu Leu35 40 45 Ile Phe Leu Asn Cys Ala Phe Thr Phe Gly Tyr Met Thr Phe Val His50 55 60 Phe Gln Ser Thr Asn Lys Val Ala Leu Thr Met Gly Ala Val Val Ala65 70 75 80 Leu Leu Trp Gly Val Tyr Ser Ala Ile Glu Thr Trp Lys Phe IleThr 85 90 95 Ser Arg Cys Arg Leu Cys Leu Leu Gly Arg Lys Tyr Ile Leu AlaPro 100 105 110 Ala His His Val Glu Ser Ala Ala Gly Phe His Pro Ile AlaAla Asn 115 120 125 Asp Asn His Ala Phe Val Val Arg Arg Pro Gly Ser ThrThr Val Asn 130 135 140 Gly Thr Leu Val Pro Gly Leu Lys Ser Leu Val LeuGly Gly Arg Lys 145 150 155 160 Ala Val Lys Gln Gly Val Val Asn Leu ValLys Tyr Lys Ala 165 170 (2) INFORMATION FOR SEQ ID NO:42: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 123 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: Met Pro Asn Asn AsnGly Lys Gln Gln Lys Lys Lys Lys Gly Asn Gly 1 5 10 15 Gln Pro Val AsnGln Leu Cys Gln Met Leu Gly Lys Ile Ile Ala Gln 20 25 30 Gln Asn Gln SerArg Gly Lys Gly Pro Gly Lys Lys Asn Lys Lys Lys 35 40 45 Asn Pro Glu LysPro His Phe Pro Leu Ala Thr Glu Asp Asp Val Arg 50 55 60 His His Phe ThrPro Ser Glu Arg Gln Leu Cys Leu Ser Ser Ile Gln 65 70 75 80 Thr Ala PheAsn Gln Gly Ala Gly Thr Cys Thr Leu Ser Asp Ser Gly 85 90 95 Arg Ile SerTyr Thr Val Glu Phe Ser Leu Pro Thr His His Thr Val 100 105 110 Arg LeuIle Arg Ala Thr Ala Ser Pro Thr Ala 115 120 (2) INFORMATION FOR SEQ IDNO:43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)ORIGINAL SOURCE: (A) ORGANISM: Synthetic (xi) SEQUENCE DESCRIPTION: SEQID NO:43: CTGAGAATTC ATGGGGGCTA TGCAAAGCCT TTTCG 35 (2) INFORMATION FORSEQ ID NO:44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Synthetic (xi)SEQUENCE DESCRIPTION: SEQ ID NO:44: GTGGAATTCA CCGTGAGTTC GAAAGAAAAATTGC 34 (2) INFORMATION FOR SEQ ID NO:45: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM:Synthetic (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: CAATGAATTC TAGCTGTGCACTCCTCCATA TTTTCCTC 38 (2) INFORMATION FOR SEQ ID NO:46: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINALSOURCE: (A) ORGANISM: Synthetic (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:CGGGAATTCC TATCGCCGTA CGGCACTGAG G 31 (2) INFORMATION FOR SEQ ID NO:47:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)ORIGINAL SOURCE: (A) ORGANISM: Synthetic (xi) SEQUENCE DESCRIPTION: SEQID NO:47: GGGAATTCTG CGTCCCTTCT TTTCCTCTTG GTTG 34 (2) INFORMATION FORSEQ ID NO:48: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Synthetic (xi)SEQUENCE DESCRIPTION: SEQ ID NO:48: CTTGGATCCT CAAATTGCCA GCAGGATGGCAAAAAG 36 (2) INFORMATION FOR SEQ ID NO:49: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINALSOURCE: (A) ORGANISM: Synthetic (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:CAAGGAATTC GGGGAAATGC TTGACCGCGG GCTG 34 (2) INFORMATION FOR SEQ IDNO:50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)ORIGINAL SOURCE: (A) ORGANISM: Synthetic (xi) SEQUENCE DESCRIPTION: SEQID NO:50: AAAAGGATCC TAGGGACGAC CCCATTGTTC AGC 33 (2) INFORMATION FORSEQ ID NO:51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Synthetic (xi)SEQUENCE DESCRIPTION: SEQ ID NO:51: CAATGAATTC GTCCCTAGAC GACTTTTGCAATGATAG 37 (2) INFORMATION FOR SEQ ID NO:52: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINALSOURCE: (A) ORGANISM: Synthetic (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:CTTGGGATCC TTATTTGGCA TATTTGACAA GGTTTACCAC 40 (2) INFORMATION FOR SEQID NO:53: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO(vi) ORIGINAL SOURCE: (A) ORGANISM: Synthetic (xi) SEQUENCE DESCRIPTION:SEQ ID NO:53: TATGAATTCT AACAACGGCA AGCAGCAAAA GAAAAAG 37 (2)INFORMATION FOR SEQ ID NO:54: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Synthetic(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: TGTGGATCCA TCACGCTGTGGGTGATGCTG TAG 33 (2) INFORMATION FOR SEQ ID NO:55: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 333 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINALSOURCE: (A) ORGANISM: Swinepox virus (xi) SEQUENCE DESCRIPTION: SEQ IDNO:55: CTGGTGCCGG AAACCAGGCA AAGCGCCATT CGCCATTCAG GCTGCGCAAC TGTTGGGAAG60 GGCGATCGGT GCGGGCCTCT TCGCTATTAC GCCAGCTGGC GAAAGGGGGA TGTGCTGCAA 120GGCGATTAAG TTGGGTAACG CCAGGGTTTT CCCAGTCACG ACGTTGTAAA ACGACGGGAT 180CTGTCATTTA TATAAATTAA TTCTAAATAA TATAGGATCC CGATCTTGTC GTATATGTTG 240TGTTATTGAA TGCGTGTTTT CTACAAACTC TTTTATGCGT AGAAACTTTT AACGTTGGTG 300TATCAAATGT CTAGAACTAG TGGATCCCCC GGG 333 (2) INFORMATION FOR SEQ IDNO:56: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)ORIGINAL SOURCE: (A) ORGANISM: Synthetic (xi) SEQUENCE DESCRIPTION: SEQID NO:56: TTGGGCGCGC CATTTATCTA TTGATCACAT CGATCAG 37 (2) INFORMATIONFOR SEQ ID NO:57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Synthetic (xi)SEQUENCE DESCRIPTION: SEQ ID NO:57: CCGGCGCGCC AGACTGCAAC AGATCTACTAGACAAGAGTC 40 (2) INFORMATION FOR SEQ ID NO:58: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 333 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINALSOURCE: (A) ORGANISM: Synthetic (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:CTGGTGCCGG AAACCAGGCA AAGCGCCATT CGCCATTCAG GCTGCGCAAC TGTTGGGAAG 60GGCGATCGGT GCGGGCCTCT TCGCTATTAC GCCAGCTGGC GAAAGGGGGA TGTGCTGCAA 120GGCGATTAAG TTGGGTAACG CCAGGGTTTT CCCAGTCACG ACGTTGTAAA ACGACGGGAT 180CTGTCATTTA TATAAATTAA TTCTAAATAA TATAGGATCC CGATCTTGTC GTATATGTTG 240TGTTATTGAA TGCGTGTTTT CTACAAACTC TTTTATGCGT AGAAACTTTT AACGTTGGTG 300TATCAAATGT CTAGAACTAG TGGATCCCCC GGG 333

What is claimed is:
 1. A recombinant raccoonpox virus comprising araccoonpox virus viral genome which contains a foreign DNA sequenceinserted into a non-essential region within the HindIII “U” genomicregion (SEQ ID NO:1) of the raccoonpox virus genome.
 2. The recombinantraccoonpox virus of claim 1, wherein the foreign DNA sequence isinserted into an O1L open reading frame (SEQ ID NO:3).
 3. Therecombinant raccoonpox virus of claim 1, wherein the foreign DNAsequence is inserted into an E11L open reading frame (SEQ ID NO:2). 4.The recombinant raccoonpox virus of claim 1, wherein the foreign DNAsequence is inserted into an intergenic region.
 5. The recombinantraccoonpox virus of claim 2, wherein the foreign DNA sequence isinserted into an XbaI restriction endonuclease site.
 6. The recombinantraccoonpox virus of claim 1, which further comprises the foreign DNAsequence inserted into the HindIII “U” genomic region, wherein theforeign DNA is capable of being expressed in a recombinant raccoonpoxvirus host cell.
 7. The recombinant raccoonpox virus of claim 1, whereinthe foreign DNA sequence encodes a screenable marker.
 8. The recombinantraccoonpox virus of claim 7, wherein the screenable marker is E. colibeta-galactosidase.
 9. The recombinant raccoonpox virus of claim 7,wherein the screenable marker is E. coli beta-glucuronidase.
 10. Therecombinant raccoonpox virus of claim 1, wherein the foreign DNAsequence encodes an antigenic polypeptide.
 11. The recombinantraccoonpox virus of claim 10, wherein the antigenic polypeptide whenintroduced into the host cell, induces production of antibodies againsta feline disease-causing agent from which the antigenic polypeptide isderived or derivable.
 12. The recombinant raccoonpox virus of claim 11,wherein the antigenic polypeptide is derived or derivable from a groupconsisting of feline pathogen, canine pathogen, equine pathogen, bovinepathogen, caprine pathogen, avian pathogen, porcine pathogen, or humanpathogen.
 13. The recombinant raccoonpox virus of claim 1, wherein theforeign DNA sequence is selected from a group consisting of swineinfluenza virus hemagglutinin, swine influenza virus neuraminidase,porcine reproductive and respiratory virus (PRRS) ORF2, PRRS ORF3, PRRSORF4, PRRS ORF5, PRRS ORF6, PRRS ORF7, porcine parvovirus VP2.
 14. Therecombinant raccoonpox virus of claim 1, wherein the foreign gene isunder control of an endogenous upstream, raccoonpox virus promoter. 15.The recombinant raccoonpox virus of claim 14, wherein the promoter isselected from a group consisting of the raccoonpox virus E11L promoterand the raccoonpox virus O1L promoter.
 16. The recombinant raccoonpoxvirus of claim 1, wherein the foreign gene is under control of aheterologous upstream promoter.
 17. The recombinant raccoonpox virus ofclaim 16, wherein the promoter is selected from a group consisting ofthe synthetic pox late promoter 1 (LP1), the synthetic pox earlypromoter 1 (EP1), the synthetic pox early promoter 2 (EP2), thesynthetic pox late promoter 2 early promoter 2 (LP2EP2), the syntheticpox late promoter 1 (LP1), the synthetic pox late promoter 2 (LP2), andthe swinepox virus I5L promoter.
 18. The recombinant raccoonpox virus ofclaim 1 designated S-RPV-002.
 19. A host cell infected with therecombinant raccoonpox virus of claim
 1. 20. A host cell of claim 19,wherein the host cell is mammalian cell.
 21. An immunologicalcomposition against an animal pathogen which comprises an effectiveimmunizing amount of the recombinant raccoonpox virus of claim 1 and asuitable carrier.
 22. A method of immunizing an animal against an animalpathogen which comprises administering to the animal an effectiveimmunizing dose of the immunological composition of claim
 21. 23. Amethod of immunizing an animal against a feline pathogen which comprisesadministering to the animal an effective immunizing dose of theimmunological composition of claim
 21. 24. The recombinant raccoonpoxvirus of claim 1, wherein the foreign DNA sequence is inserted into anintergenic region. claim 21.